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Expression, Purification and Characterization ofFungal and Viral
Recombinant Proteins
Helena Vihinen
Department of Chemical TechnologyHelsinki University of
Technology
andInstitute of Biotechnology
University of HelsinkiFinland
Dissertation for the degree of Doctor of Science in Technology
to be presented with duepermission of the Department of Chemical
Technology for public examination and debatein Auditorium KE1 at
Helsinki University of Technology (Espoo, Finland) on the 22nd
of March, 2001, at 12 noon.
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SUPERVISED BY:
Professor Leevi KääriäinenInstitute of Biotechnology, University
of Helsinki
andProfessor Marja Makarow
Institute of Biotechnology, University of Helsinki
REVIEWED BY:
Docent Anu JalankoDepartment of Human Molecular Genetics
National Public Heath InstituteHelsinki
andDocent Johan Peränen
Institute of Biotechnology, University of Helsinki
OPPONENT:
Docent Pertti MarkkanenBiotechnology Program
Espoo-Vantaa Institute of Technology
ISSN 1239-9469ISBN 951-45-9874-1
ISBN 951-45-9875-X (pdf)Gummerus Kirjapaino Oy
Saarijärvi 2001
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To my brother Harri
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CONTENTSAbbreviationsOriginal
publicationsABSTRACT...........................................................................................................................1INTRODUCTION.................................................................................................................2
1. PRODUCTION SYSTEMS FOR RECOMBINANT PROTEINS
......................... 21.1. BACTERIAL EXPRESSION UTILIZING E.
COLI
..........................................................3
1.1.1. Expression systems
......................................................................................31.1.2.
Solubility......................................................................................................41.1.3.
Secretion
......................................................................................................5
1.2. YEAST (SACCHAROMYCES CEREVISIAE)
...............................................................51.2.1.
Episomal vectors and chromosomal integration
..........................................51.2.2. Promoter
strength.........................................................................................61.2.3.
Secretion of heterologous
proteins...............................................................7
1.2.3.1. Yeast secretory proteins as carriers
.....................................................71.2.3.2. The
secretion pathway of S. cerevisiae
...............................................8
1.2.4. Optimizing expression
.................................................................................91.3.
INSECT CELLS (BACULOVIRUS
SYSTEM)..............................................................10
1.3.1. Systems utilizing polyhedrin and
p10........................................................111.3.2.
Recombination
...........................................................................................111.3.3.
Optimizing expression
...............................................................................12
1.4. MAMMALIAN EXPRESSION SYSTEMS UTILIZING VACCINIA AND
ALPHAVIRUS VECTORS
.........................................................................................131.4.1.
Vaccinia virus vector
.................................................................................131.4.2.
Alphavirus vectors
.....................................................................................14
1.4.2.1. Semliki Forest virus
..........................................................................141.4.2.1.1.
Nsp1..........................................................................................151.4.2.1.2.
Nsp2..........................................................................................161.4.2.1.3.
Nsp3..........................................................................................161.4.2.1.4.
Nsp4..........................................................................................17
2. PURIFICATION STRATEGIES FOR RECOMBINANT
PROTEINS.....................172.1. CONVENTIONAL CHROMATOGRAPHY
..................................................................17
2.1.1. Ion exchange
chromatography...................................................................182.1.2.
Reversed phase chromatography
...............................................................182.1.3.
Gel permeation
chromatography................................................................19
2.2. AFFINITY
CHROMATOGRAPHY.............................................................................192.2.1.
Affinity tags
...............................................................................................202.2.2.
Cleavage.....................................................................................................23
3. ANALYSIS OF POSTTRANSLATIONAL MODIFICATIONS
..............................243.1.
GLYCOSYLATION.................................................................................................253.2.
ACYLATION.........................................................................................................253.3.
PHOSPHORYLATION.............................................................................................26
AIMS OF THE STUDY
.....................................................................................................28MATERIALS
AND METHODS
.......................................................................................29
1.
MATERIALS................................................................................................................291.1.
STRAINS, CELLS AND
MEDIA................................................................................291.2.
PLASMID CONSTRUCTIONS
..................................................................................30
1.2.1. Yeast (I-III)
................................................................................................301.2.2.
E. coli (IV,
unpublished)............................................................................301.2.3.
Baculovirus system
(unpublished).............................................................30
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1.2.4. Vaccinia infection/DNA-transfection (IV-
IV)..........................................302. METHODS
...................................................................................................................30
2.1. DELETION CONSTRUCTS AND POINT MUTATIONS (IV-VI)
...................................302.2. IMMUNOPRECIPITATION,
IMMUNOBLOTTING AND SDS-PAGE ...........................302.3.
PROTEIN EXPRESSION AND PURIFICATION
...........................................................31
2.3.1. Yeast (I-III)
................................................................................................312.3.2.
E.coli (IV,
unpublished).............................................................................312.3.3.
Sf9 cells (unpublished)
..............................................................................322.3.4.
HeLa cells
(IV-VI).....................................................................................32
2.4. METABOLIC LABELING
(I-III,V,VI).....................................................................342.5.
IMMUNOFLUORESCENCE
(VI)..............................................................................342.6.
FLOTATION ANALYSIS (VI)
.................................................................................342.7.
PHOSPHOAMINO ACID ANALYSIS
(V)...................................................................342.8.
PHOSPHOPEPTIDE MAPPING
(V)...........................................................................352.9.
OTHER SPECIFIC METHODS
..................................................................................35
RESULTS
............................................................................................................................36
1. HSP150� AS A CARRIER FOR SECRETION OF RECOMBINANT PROTEINS IN
S. cerevisiae
.............................................................................................................36
1.1. CHARACTERIZATION OF HSP150�-CARRIER
(I)...................................................361.1.1.
Expression of truncated Hsp150 (I)
.......................................................................
371.1.2. Purification of truncated and authentic Hsp150 (I)
................................................ 371.1.3.
Structural features (I)
.............................................................................................
38
1.2. SECRETION AND AUTHENTICITY OF HSP150�-NGFRe
(II)...................................381.2.1. Expression of
Hsp150�-NGFRe (II)
......................................................................
381.2.2. Purification of Hsp150�-NGFRe (II)
......................................................................
39
1.3. GLYCOSYLATION OF NGFRe (III)
.......................................................................
392. EXPRESSION, PURIFICATION AND PROPERTIES OF SFV Nsps
.......................41
2.1. EXPRESSION AND PURIFICATION OF NSPS (UNPUBLISHED)
..................................412.1.1. Expression of Nsp1 in E.
coli (unpublished)
.......................................................... 412.1.2.
Expression of N-terminal fragment of Nsp2 in E. coli
(unpublished)..................... 422.1.3. Expression of Nsp3 in
E. coli (unpublished)
.......................................................... 422.1.4.
Expression of Nsp4 in Sf9 cells (unpublished)
....................................................... 44
2.2. CHARACTERIZATION OF NSP1 AND NSP3
(IV-VI)...............................................452.2.1.
Critical residues for enzymatic activities of Nsp1
(IV)........................................... 452.2.2.
Phosphorylation of Nsp3 (V and VI)
......................................................................
46
2.2.2.1. Determination of phosphorylation sites (V)
.................................................. 462.2.2.2.
Phosphorylation of Nsp3 derivatives (VI)
..................................................... 472.2.2.3.
Elimination of phosphorylation sites (V and VI)
........................................... 482.2.2.4. Effect of
phosphorylation on membrane association (VI)
............................. 482.2.2.5. Phosphorylation deficient
Nsp3 in the context of SFV (VI) .......................... 48
DISCUSSION
......................................................................................................................491.
HSP150� AS A CARRIER FOR PROTEIN PRODUCTION
.....................................502. EXPRESSION AND
CHARACTERIZATION OF SFV
Nsps....................................50
2.1. EXPRESSION AND PURIFICATION OF
Nsps............................................................492.2.
GT AND MT ACTIVITIES OF Nsp1
.......................................................................522.3.
PHOSPHORYLATION OF
Nsp3...............................................................................52
2.3.1. Determination of phosphorylation sites
.....................................................532.3.2.
Kinases.......................................................................................................532.3.3.
Effects on the virus cycle and neurovirulence
...........................................54
3. CONCLUSIONS AND FUTURE ASPECTS
..............................................................55ACKNOWLEDGEMENTS
...............................................................................................57REFERENCES....................................................................................................................58
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ACN acetonitrileAcMNPV Autographa californica
multiple nuclear polyhedrosisvirus
AdoHcy S-adenosyl-L-homocysteineAdoMet
S-adenosyl-L-methionineBVS baculovirus expression systemBHK baby
hamster kidneyBSA bovine serum albuminCD circular dichroismCHX
cycloheximideCIAP calf intestine alkaline
phosphataseCID collision-induced dissociationCPVI cytopathic
vacuoles type IDOC deoxycholateDTT dithiothreitolECL enhanced
chemiluminescenceER endoplasmic reticulumESI electrospray
ionizationFSC fetal calf serumGnd-HCl guanidinium hydrochlorideGPC
gel permeation chromatographyGST glutathione-S-transferaseGT
guanylyltransferaseHPLC high performance liquid
chromotographyHsp heat shock proteinIEC ion exchange
chromatographyIMAC immobilized metal affinity
chromatographyIPTG isopropyl-ß-D-thiogalacto-
pyranosidekDa kilodaltonMALDI matrix-assisted laser desorp-
tion/ionization
MBP maltose binding proteinm.o.i. multiplicity of infectionMS
mass spectrometryMT methyltransferaseMVA modified vaccinia virus
AnkaraNGFRe ectodomein of rat nerve growth
factor receptorNMR nuclear magnetic resonanceNsp nonstructural
proteinNTA nitrilotriacetic acidODV occlusion derived virusPBS
phosphate buffered salinePDI protein disulfide isomerasePFU plaque
forming unitp.i. post infectionPMSF phenylmethylsulfonyl
fluoridePPM phosphopeptide mappingPVDF polyvinylidene difluorideRPC
reversed phase chromatographySC synthetic completeSf9 Spodoptera
frugiperda insect
cell lineSFV Semliki Forest virusSIN Sindbis virusSRP signal
recognition particleTCA trichloroacetic acidTGN trans Golgi
networkTLC thin layer chromatographyTLE thin layer
electrophoresisTM tunicamycinTOF time-of-flightTrx thioredoxinwt
wild type
Abbreviations
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Jämsä, E., Holkeri, H., Vihinen, H., Wikström, M., Simonen, M.,
Walse, B.,Kalkkinen, N., Paakkola, J. and Makarow, M. (1995).
Structural features of apolypeptide carrier promoting secretion of
a ß-lactamase fusion protein in yeast.Yeast 11, 1381-1391.
Simonen, M., Vihinen, H., Jämsä, E., Arumäe, U., Kalkkinen, N.
and Makarow,M. (1996). The hsp150�-carrier confers secretion
competence to the rat nervegrowth factor receptor ectodomain in
Saccharomyces cerevisiae. Yeast 12, 457-466.
Holkeri, H., Simonen, M., Pummi, T., Vihinen, H. and Makarow, M.
(1996).Glycosylation of rat NGF receptor in the yeast Saccharomyces
cerevisiae. FEBSLett. 383, 255-258.
Ahola, T., Laakkonen, P., Vihinen, H. and Kääriäinen, L. (1997).
Critical Residuesof Semliki Forest virus RNA capping enzyme
involved in methyltransferase andguanylyltransferase-like
activities. J. Virol. 71, 392-339.
Vihinen, H. and Saarinen, J. (2000). Phosphorylation site
analysis of SemlikiForest virus nonstructural protein 3. J. Biol.
Chem. 275, 27775-27783.
Vihinen, H., Ahola, T., Tuittila, M., Merits, A. and Kääriäinen,
L. (2001).Elimination of phosphorylation sites of Semliki Forest
virus replicase proteinnsP3. J. Biol. Chem. 276, in press.
I
II
III
IV
V
VI
Original publications
This thesis is based on the following original publications
which are referred to in the text bytheir Roman numerals.
Additional unpublished data will also be presented in the text.
All articles are reprinted by kind permission from the
publishers.
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1
This work reports the production of recom-binant yeast and viral
proteins in a numberof diverse in vivo model systems for enzy-matic
and structural studies. In the first partHsp150� peptide, a
derivative of the yeast(Saccharomyces cerevisiae) secretory
heat-shock protein Hsp150, was investigated forits ability to act
as a carrier in transportingthe ectodomain of rat nerve growth
factor(NGFRe) out from the yeast cell. TheHsp150�-NGFRe fusion
protein was effi-ciently secreted into the growth medium,where it
constituted the majority of totalsecreted proteins. Inhibition
experiments withpurified Hsp150�-NGFRe showed thatHsp150� did not
prevent NGFRe from fold-ing into a ligand-binding conformation.
Cir-cular dichroism (CD) analysis revealed thatthe Hsp150�-carrier
did not have any spe-cific secondary structure, which was also
sug-gested by NMR analysis of a syntheticpolypeptide corresponding
to the repetitiveconsensus sequence of subunit II of Hsp150.These
findings suggest that Hsp150� can suc-cessfully act as a carrier
for foreign proteins,such as NGFRe, made and secreted by
S.cerevisiae.
The second part of this study involved theexpression and
purification of an RNA ani-mal virus, Semliki Forest virus
(SFV),nonstructural proteins (Nsp1-4) using a num-ber of in vivo
protein expression systems. Toensure quantities large enough for
structuraland enzymatic studies of the Nsps, each ofthem was
expressed either in bacteria (Es-cherichia coli) or in insect cells
(Sf9). Allthe proteins were expressed in high quanti-ties (10-100
mg/l), and purified by affinity
and size exclusion chromatography undernondenaturing or
denaturing conditions. In-dependent of the expression system used,
allthe partially purified Nsps aggregated andprecipitated either
upon concentration, dialy-sis, storing or thawing. No detergents
werefound that could alleviate the aggregationproblem or assist in
the purification process.
Despite the unsuccessful purification ofNsps for structural
studies, the expression andpartial purification of Nsp1 and Nsp3
permit-ted biochemical characterization of their en-zyme activities
and posttranslational modifi-cations. Point mutational analysis of
the Nsp1methyltransferase domain revealed that resi-due His38 was
essential for the guanylyl-transferase activity of Nsp1.
Furthermore,residues Asp64 and Asp90 were found to beimportant for
the methyltransferase activityof Nsp1. Phosphorylation sites in
Nsp3 weredeterminated by point mutational analysis,electrospray
ionization (ESI) and matrix as-sisted laser desorption ionization
(MALDI)mass spectrometry (MS) as well as byphosphopeptide mapping
and Edman se-quencing. A phosphorylated domain (aa 320-368) was
located in the C-terminal, non-con-served region of Nsp3, where 12
serines and4 threonines could be modified by phos-phates. The
phosphorylation of Nsp3 seemednot to affect the membrane
association or thelocalization of Nsp3 in either transfected
orinfected cells. Furthermore, Nsp3 phosphory-lation deficient
mutant viruses were capableof replication in infected mammalian
cells asimilar manner to the wild type SFV, but
theirneuropathogenicity in adult mice was greatlyreduced.
ABSTRACT
Abstract
-
Introduction
2
Molecular biology offers technologieswhereby proteins can be
produced and puri-fied easier and more efficiently than everbefore.
Using recombinant DNA techniquessuch as gene fusion it is possible
to generatechimeric proteins, which are novel in struc-ture and
function. At the DNA level, aminoacid coding regions are now
routinely recom-bined to incorporate new functional
domains,proteolytic cleavage sites, intra- and extra-cellular
localization or targeting signals,stabilizing signals, and even
amino acidsequences that facilitate protein purification.All of
these directed mutations can be used tosubtly or dramatically alter
the properties (e.g.,solubility, electric charge,
hydrophobicity,conformation, substrate binding and activesites) of
the protein. Thus, protein engineer-ing has become a powerful tool
in molecularbiology to investigate protein structure andfunction,
in addition to production and purifi-cation of useful proteins
(Sassenfeld, 1990).The main applications of recombinant
proteinsobtained by genetic engineering are in themedical
therapeutic fields (e.g., production ofrecombinant vaccines, and
therapeutic proteinsfor human diseases), and medical
diagnosis(e.g., antigen engineering for poly- and mono-clonal
antibody production used in diseasetesting). Other areas where
recombinant pro-teins are commonly utilized include enzymesfor food
and fiber production, testing food formicrobial contamination and
veterinary medi-cine (Nilsson et al., 1992).
Besides producing valuable proteins forpractical applications,
the production ofproteins using recombinant technology
offersunprecedented possibilities in the basicresearch of protein
function, structure, inter-action, turnover, domain shuffling, and
manyother areas, which could ultimately lead to thedevelopment of
novel protein applications.Most proteins are expressed in
infinitesimal
amounts in their native cells and tissues, andit is only by
recombinant techniques that it ispossible to produce amounts great
enough forbasic research or for practical uses. Therefore,the
expression of engineered proteins in effi-cient heterologous
protein expression systemis integral to the production, and
purificationof many proteins of interest. Moreover, withthe vast
amounts of predicted protein se-quences being generated by genomic
research,the application of protein engineering will mostcertainly
be used to better characterize manyunknown protein functions,
interactions,cellular locations and potential practical uses.
1. PRODUCTION SYSTEMS FORRECOMBINANT PROTEINS
Although much of recombinant proteinengineering is performed in
vitro, the actualprotein synthesis usually utilizes
livingprokaryotic or eukaryotic cells. Proteins pro-duced in vivo
can be expressed transiently,constitutively or by induction, with
the last tworequiring cell lines which stably maintain thegenetic
material coding for the protein.Another means consist of using live
bio-reactors, i.e., live transgenic plants andanimals, as
recombinant protein productionsystems. The choice of production
systemdepends on many factors including the origi-nal source of the
protein and codon usage (e.g.,prokaryotic, mammalian, plant),
posttransla-tional processing (e.g., phosphorylation, pro-teolytic
processing, glycosylation, acylation),its future use (e.g., human
therapy, plant pestcontrol) and economic factors (Hodgson,1993).
However, production of any protein isa complex multistep cellular
process involv-ing gene transcription, translation,
proteinprocessing and localization. Thus, the expres-sion of a
recombinant protein in a heterolo-gous host cell or organism is
often problem-atic. For example, protein expression levels
INTRODUCTION
-
Introduction
3
can be low, or the purified protein may beinsoluble or unstable.
And although, certainprecautions can be taken (e.g., altering
codonusage to match the host’s cells), often trial anderror is the
only real method of operation.
1.1. BACTERIAL EXPRESSION UTILIZING E. COLI
Escherichia coli (E. coli) has long been theprimary prokaryotic
host for heterologousprotein expression, therefore there is
muchinformation and technology regarding thissystem with many
different proteins (Baneyx,1999; LaVallie et al., 1995). The
advantagesof E. coli include its relatively easy, rapid
andinexpensive methods of growth, transforma-tion and maintenance.
Furthermore, most ofthe biochemical pathways of E. coli
areunderstood in great detail, and its entiregenome has been
sequenced. Foreign proteinscan be produced in E. coli in large
amounts(5-50% of total protein). Nevertheless,prokaryotic cells
such as E. coli are unable toperform some posttranslational
modifications,which occur in eukaryotic cells (e.g.,
glyco-sylation, phosphorylation and disulfidebondformation), and
which may be crucial for aforeign protein to obtain an active form.
Theexpression and accumulation of a foreign pro-tein in E. coli may
also cause aggregates ofthe protein to form (Lilie et al., 1998;
DeBerbardez Clark, 1998). Sometimes theprotein in these inclusion
bodies can berefolded in vitro to produce functional protein,but
this is not always possible and renaturationcan be expensive and
time consuming. Fur-thermore, proteolytic degradation of
heterolo-gous protein expression may be a potentialobstacle (Murby
et al., 1996; Matsuo et al.,1999). Even though E. coli may not be
usefulfor all foreign protein production, it has beensuccessfully
utilized to produce many func-tional human proteins such as human
growthhormone, proinsulin, interferon-gamma andantibody fragments
(Patra et al., 2000; Cowleyand Mackin, 1997; Davis et al.,
1999;Plückthun, 1992).
1.1.1. Expression systems
Various E. coli expression systems areavailable commercially and
in the publicdomain, which is shared within the
scientificcommunity. Each system offers differentbenefits for
protein expression, detection, andpurification, and should be
consideredaccording to the specific criteria and require-ments each
protein poses. The expression unitor cassette in which the foreign
protein codingregion will be cloned into should consist of astrong
promoter (e.g., T7, trc, lac, tac, PL, PR,phoA, ara, xapA, cad,
recA), ribosomalbinding site(s), and an efficient
transcriptionterminator (LaVallie et al., 1995). Tightlycontrolled
transcription coupled with a strongand rapid transcription inducer
(e.g., IPTG,tryptophan, temperature, phosphatase starva-tion,
arabinose, xanthosine, pH, nalidixic acid)are crucial for high
protein expression. Otherfactors to be considered include
convenientmultiple cloning sites, appropriate codonusage, and
plasmid copy number. Usually, highprotein expression levels are
desirable, but incases where the protein may form
insolubleinclusion bodies, or may be toxic to the bacte-rial host,
lower and slower expression may bepreferred. The ideal promoter for
expressiondirects efficient transcription to allow high-level
production, and is tightly regulated tominimize the metabolic
burden and toxiceffects of a foreign protein. Regulatable
pro-moters are used to drive foreign proteinexpression to avoid the
selection of non-expressing mutant cells during the cell
growthphase. Promoters such as T3, SP6 and T7 arewidely used and
efficient promoters derivedfrom E. coli bacteriophages.
The T7 RNA polymerase is very specificfor its own promoter, and
can transcribe RNAtemplates approximately five times faster thanthe
E. coli RNA polymerase (Mertens et al.,1995). The gene coding for
T7 RNA poly-merase is either lysogenic or it is transfectedinto the
cell by the bacteriophage CE6. In DE3E. coli cells the T7 RNA
polymerase gene is
-
Introduction
4
under the control of the LacUV5 promoter,which is inducible by
IPTG. The addition ofIPTG to a culture of DE3 cells will induce
thetranscription of T7 RNA polymerase, whichin turn transcribes any
gene under the controlof the T7 promoter. However, the basal
levelof T7 RNA polymerase activity will promotesome transcription
of the target gene in non-induced cells. More strict control
mechanismshave been engineered to depress basal T7 RNApolymerase
expression. One method is basedon the addition of a regulatory lacI
gene, whichcodes for a strong repressor of the LacUV5promoter
(Dubendorff and Studier, 1991). Verylow basal expression levels of
target gene canbe reduced even more by adding T7 lysozyme,yet high
level expression can be achieved uponinduction with IPTG. Moreover,
the T7lacOexpression vectors pBAT, pHAT and pRAThave been reported
to be suitable for expres-sion of toxic proteins in E. coli
(Peränen etal., 1996). In these vectors tight control isachieved by
centering the lac operator 14 basepairs downstream from the major
RNA tran-scription start point.
1.1.2. Solubility
The majority of foreign proteins producedin E. coli are
cytosolic, but as mentioned abovesome proteins accumulate as
aggregates calledinclusion bodies. In these aggregates only asmall
fraction of the protein is folded correctly,and the rest is in a
denatured and usually non-functional form (Geisow, 1991; Valax
andGeorgiou, 1993). The probability of any het-erologous protein
forming inclusion bodiescorrelates greatly with its charge average
andturn forming residue fraction, whereas hydro-philicity and total
number of residues do notappear to correlate with inclusion body
for-mation (Wilkinson and Harrison, 1991).Inclusion bodies may
consist of not only theforeign protein, but also of some host
compo-nents such as T7 RNA polymerase, membranecomponents, 16S and
23S RNA and even plas-mid DNA (Mitraki and King, 1989). In
somecases the formation of inclusion bodies can be
advantageous, since the expressed protein canbe purified simply
by washing and pelletingthe aggregated bodies (Georgiou and
Valax,1999). However, often the aggregation of theprotein is
detrimental, therefore expressionconditions must be optimized so
that thepossibility of producing soluble protein isincreased. The
maximized solubility of aprotein can be achieved by optimizing
severalfactors such as the expression temperature, E.coli strain
used, pH of the growth medium,fusion partner the protein is fused
with, rateand level of expression and co-expression withchaperones
and foldases (Murby et al., 1996;Zhang et al., 1998; Weickert et
al., 1996). Forexample, fusions to ZZ (two domains derivedfrom
staphylococcal protein A) and thio-redoxin have been reported to
increase thesolubility of insulin-like growth factor I andcytokines
(Samuelsson et al., 1994; LaVallieet al., 1993). Chaperone proteins
mediate boththe folding and rehabilitation of proteins (vanDyk et
al., 1989; Bukari and Zipser, 1973).The most extensively studied
chaperons/foldases in E. coli are GroEL/GroES, whichhave been
reported to assist in folding proteinsinto active forms
(Goloubinoff et al., 1989;Lee and Olins., 1992; Yasukawa et al.,
1995;Amrein et al., 1995; Wall and Plückthun,1995). Moreover,
co-overproduction ofthioredoxin has been reported to increase
thesolubility of insulin-like growth factor I as wellas other
vertebrate proteins expressed in E. coli(Yasukawa et al., 1995).
However, even if co-overexpression of chaperons/foldases
(e.g.,GroES, GroEL, DnaK) is likely to be usefulfor some proteins,
it is unlikely to provide auniversal solution to the problem of
inclusionformation (Hockney, 1994). Other factors af-fecting
protein solubility include regulation ofthe expression vector and
bacterial cell lysismethods. Protein inclusion bodies are
usuallysolubilized using urea or guanidine hydro-cloride (Gdn-HCl).
After denaturation the pro-tein must be renatured under conditions
thatfavor their proper folding and native functions.Conditions that
must be considered when
-
Introduction
5
trying to refold a denatured protein involveoptimizing the
renaturation temperature(usually around 10°C), redox
conditions,protein concentration, and addition of solubi-lizing
additives such as polyethylene glycol.Soluble and active proteins
have also beenobtained with treatment with sarkosyl
(N-dodecanoyl-sarcosinate, Frangioni and Neel,1992).
1.1.3. Secretion
Secretion of foreign proteins to the mediumcan greatly
facilitate their purification.However, secretion of a protein to
the mediumby Gram-negative bacteria, such as E. coli,requires
translocation of the protein throughthe bacterial cell wall
consisting of inner andouter membrane bilayers surrounding the
peri-plasmic space. Only a limited number of E.coli strains are
capable of secreting proteinsfrom the cell (reviewed in Pugsley,
1993).Nevertheless, some genetically modifiedproteins with inserted
secretion sequence sig-nals, have been successfully secreted into
themedia (H�gset et al., 1990; Wadensten et al.,1991; Weiss et al.,
1994; Samuelsson et al.,1994). More importantly, E. coli is capable
ofsecreting genetically altered proteins into theperiplasm, which
may represent 20-40% of thetotal cellular volume. With this
strategy thetarget protein can be separated from the cyto-plasmic
proteins and accumulate in a moreoxidizing environment where
disulfide-bondformation can occur (Missiakas et al.,
1993).Secretion into the periplasmic space has beensuccessfully
utilized in large scale productionof biologically active antibody
fragments(Plückthun, 1992).
1.2. YEAST (SACCHAROMYCES CEREVISIAE)Saccharomyces cerevisiae
(bakers and brew-
ers yeast) is generally considered as a safe or-ganism for the
production of foreign proteins,since it has been used in food
production forthousands of years (Müller et al., 1998).
Thisunicellular eukaryote has been extensively
studied at the biochemical, molecular andgenomic levels, and its
entire genome has beenrecently sequenced (Goffeau et al., 1997).
LikeE. coli, yeast grows rapidly in relatively in-expensive and
simple media, is easy to trans-form, and maintain (Buckholz and
Gleeson,1991). But unlike E. coli, yeast is an eukaryoticcell, and
therefore expresses and processes pro-teins more similarly to
higher eukaryotes. (LinCereghino and Cregg, 1999). The
secretorypathway of yeast closely resembles that ofmammalian cells,
thus yeast is capable of manyposttranslational modifications such
as pro-teolytic processing, disulfide bond formation,and
glycosylation (Eckart and Bussineau,1996). Thus, yeast offers a
eukaryotic proteinproduction system that can retain the
foreignprotein intracellularly or the product can besecreted to the
medium if fused to a carrierpeptide. However, S. cerevisiae is
unable toperform certain complex eukaryotic posttrans-lational
modifications such as prolylhydroxy-lation and amidation. In
addition, the glyco-sylation of yeast can differ from that of
highereukaryotes (Sudbery, 1996). Nevertheless,many foreign
proteins have been produced ona large scale using S. cerevisiae,
includinghuman serum albumin, the hepatitis B viralsurface antigen,
insulin and hirudin (Goodly,1993; Valenzuela et al., 1982; Thim et
al.,1986; Ladisch and Kohlmann, 1992; Mendoza-Vega et al.,
1994).
1.2.1. Episomal vectors and chromosomalintegration
Recombinant DNA which encodes foreignproteins can be expressed
in S. cerevisiae asan episomal plasmid or integrated into
itsgenome. Extra-chromosomal replicons arebased either on plasmids
containing auto-nomously replicating sequences, or on the na-tive
2µ circle of S. cerevisiae (Romanos et al.,1992). Most yeast
expression vectors havebeen constructed from the multi-copy
2µplasmid, which has also been engineered toreplicate and be
selectable in both E. coli and
-
Introduction
6
yeast. Because the native 2µ circle is presentin most S.
cerevisiae strains at approximately60-100 copies per haploid
genome, and theplasmid is stable inherited, 2µ-based
proteinproduction can be very efficient (Futcher,1988; Unternährer
et al., 1991). The most com-mon selection markers are LEU2, TRP1,
URA3and HIS3 in corresponding amino acid (aa)uptake deficient
mutant strains, which areauxotrophic for leucine, tryphophan,
uracil andhistidine, respectively. Selection can also beperformed
using e.g., the antibiotic resistanceelement G418, a
2-deoxy-streptamine antibi-otic (Jimenez and Davies, 1980). There
arealso yeast strains engineered to ensure plasmidmaintenance
irrespective of the culture condi-tions, for example, ura3 fur1
-strains are non-viable on uracil minus media since they areblocked
both in the de novo and salvage path-ways of uridine
5’-monophosphate synthesis(Loison et al., 1986; Napp and Da Silva,
1993).These strains can grow on uracil minus mediaonly if they
maintain a plasmid that containsthe URA3 gene, which complements
themutant allele.
Chromosomal integration offers a morestable alternative to
episomal maintenance ofplasmid DNA coding for foreign proteins.
Themost widely utilized technique to obtain inte-gration is
homologous recombination (re-viewed in Romanos et al., 1992).
Integrationvectors are similar to the above mentionedyeast plasmids
with both E. coli and yeastselectable markers and bacterial
replicationsequences, but lack yeast replication se-quences. Once
the plasmid has been trans-formed into a yeast cell, usually in a
linearform, it can not replicate, and is maintainedonly if it
integrates into the yeast genome.Homologous recombination can occur
by asingle or double crossover, either as a multi-copy integration
into ribosomal DNA, or bytransposable elements such as Ty or
gamma(Fleer, 1992). Double cross-over vectors con-tain the foreign
DNA and selection markerflanked by yeast DNA homologues to the
5’and 3’ regions of the chromosomal DNA to
be replaced. The frequency of transformationby this method is
lower than episomal plasmidtransformation, but it results in very
stable,usually single copy transformants (Romanoset al., 1992).
Multicopy integration of heter-ologous genes usually employs
integration intoreiterated chromosomal DNA sequences thatmay be
present from 20 to 140 repeats perhaploid genome. Chromosomal
integrationcan also be obtained by using Ty transposi-tion vectors,
which are analogous to theretroviral vectors of mammalian cells
(Boekeet al., 1988).
1.2.2. Promoter strength
Many different yeast transcriptional promot-ers are available
for foreign protein expres-sion in S. cerevisiae (Fleer 1992).
Yeastpromoters consist of at least three elementswhich regulate the
efficiency and accuracy ofthe initiation of mRNA transcription.
Theseelements are upstream activation sequences(UAS), the TATA-box
and initiator elements(Romonos et al., 1992). Numerous strongyeast
promoters to be utilized are either in-ducible (e.g., GAL1, GAL7,
GAL10, MET25),or constitutive (e.g., CYC1, ADH1, TEF2,GPD and MF�)
(Mumberg et al., 1994 and1995). In the case of large scale
proteinproduction in yeast, it is crucial to be able toseparate the
cell growth phase from the proteinproduction phase (Da Silva and
Bailey, 1991).The promoter strength of three different pro-moters
i.e., SUC2, PGK and GAL7 were stud-ied when �-amylase was produced
in 30 hourfed-batch yeast culture (Park et al., 1993).According to
the results the efficiency of thepromoters appeared to be in the
followingorder PGK> GAL7> SUC2, while plasmidstability and
promoter strength appeared to beinversely correlated. Also
expression vectorswith a tetracycline and copper ion
regulatablepromoter systems have been developed (Gariet al., 1997;
Labbé and Thiele, 1999). Anotherapproach to optimize gene
expression in yeastutilizes transactivators such as
mammaliansteroid hormone receptors, which have been
-
Introduction
7
reported to increase transcriptional activity(Fleer, 1992), as
well as regulate the overpro-duction of the GAL4 protein (Schultz
et al.,1994).
1.2.3. Secretion of heterologous proteins
S. cerevisiae is capable of secreting heter-ologous proteins,
but most of them have to befused to an appropriate
pre-pro-leader(Romanos et al., 1992; Shuster, 1991). Thesecarriers
are relatively short amino acid se-quences and include
pre-pro-leaders from suchproteins as �-factor, killer-toxin, bar
protease,gp37, and Hsp150 (reviewed by Simonen,1994). The carrier
peptide may have a crucialrole in the transport process from the ER
tothe Golgi, which has been suggested to be re-ceptor- mediated
(Balch et al., 1994). The car-rier may thus provide a positive
secretion sig-nal for the capture of fusion protein into
vesiclebudding sites at the ER membrane (Suntio etal., 1999). The
carrier may also assist the fu-sion partner to acquire a
secretion-competentconformation (Simonen et al., 1994; Simonenet
al., 1996; Hammond and Helenius, 1995).
Some proteins can be externalized in S.cerevisiae without a
carrier. Human lipo-cor-tin-1 was first expressed as a fusion with
the
�-factor pre-pro-region but only 10% of theprotein was secreted
(Chung et al., 1996). Anexpression system utilizing the inulinase
sig-nal peptide increased secreted lipocortin-1 5-fold, resulting
up to 95% of the protein to besecreted into the medium (Chung et
al., 1996).Furthermore, fed-batch fermentation of theheterologous
protein produced 2.1 g/l, repre-senting more than 80% of the total
extracellu-lar protein (Chung et al., 1999). Also, it hasbeen
observed that culture growth conditionscan affect protein secretion
(Rossini et al.,1993). For example, ß-galactosidase, which
isnormally refractile to secretion, is secretedfrom yeast grown in
a rich medium. ß-galac-tosidase secretion could be further
increasedby elevated growth temperatures.
1.2.3.1. Yeast secretory proteins ascarriers
In S. cerevisiae the most commonly used car-rier is the
pre-pro-region of the �-factor (Fig.1A and 1B). The pre-sequence
(signal se-quence) is cleaved upon translocation into theER and the
pro-region is cleaved by the Kex2protease in the Golgi compartment
(Julius etal., 1984). However, the mechanisms whichthe �-factor
pre-pro-region uses to confer
Figure 1. A Schematic presentation of primary translation
products of S. cerevisiae mating �-factor(A), pre-pro-region of
�-factor used as a carrier (B), killer toxin (C) and pre-pro-region
of killer toxinused as a carrier (D). Pre-pro-killer toxin carrier
comprises killer toxin residues 1-34, which are joinedby alanine to
the gamma-region residues 177-233. The Kex2 cleavage and
N-glycosylation sites areindicated by arrows and asterisks,
respectively. The signal sequence is marked by a horizontally
stripedarea and the repetitive region of �-factor is colored
gray.
-
Introduction
8
secretion competence to the fusion partners,as well as its
structure, are not known. Syn-thetic leaders have also been used as
carriers(Kjeldsen et al., 1997 and 1998b). Interest-ingly, it
appears that three N-linked oligosac-charide chains are necessary
for secretion com-petence of the �-factor pre-pro-leader,
whereassynthetic pre-pro-leaders lacking the consen-sus
N-glycosylation sites confers secretioncompetence of correctly
folded insulin precur-sors (Kjeldsen et al., 1998a and 1998b).
Another polypeptide used as a carrier inyeast is derived from
the killer toxin (Fig. 1D).The K1 killer toxin is a heterodimer of
twoprotein subunits (� and ß), maturated from the316 residue
pre-pro-toxin (Fig. 1C) (Bostianet al., 1984). As in the
pre-pro-region of �-factor, the signal sequence of the
pre-pro-toxinis cleaved upon translocation into the ER andthe
pro-region is subsequently cleaved by theKex2 protease in the Golgi
compartment(Redding et al., 1991). The ability of
differentfragments of the killer toxin pre-pro-region toconfer
secretion competence for heterologousprotein was studied by fusing
ß-lactamase tovarious fragments of pre-pro-toxin (Cartwrightet al.,
1992). The most efficiently secreted con-struct had a carrier in
which the genes of theß-subunit and the control region of �
weredeleted (Fig. 1D). Secreted ß-lactamasereached level of several
µg/ml which is equalto the level produced by the pre-pro-region
ofthe �-factor (Cartwright et al., 1994).
1.2.3.2. The secretion pathway of S.cerevisiae
The secretory pathway in eukaryotic cellsis essential for
posttranslational processing,targeting and transporting proteins to
intra- andextracellular membranes and for proteins des-tined for
export out of the cell. The secretorypathway of yeast is depicted
in Figure 2. Itconsists of several membrane-bound compart-ments and
vesicle-trafficing systems betweenthese compartments. Protein
secretion in yeasthas been studied using
temperature-sensitivesecretion (sec) mutants, in which the
transport
of proteins can be reversibly blocked in dis-tinct compartments
(Kaiser and Schekman,1990). Secretery proteins have a signal
pep-tide, which is an endoplasmic reticulum (ER)targeting sequence
present usually at their N-terminus, which allows the protein to
enter theER. Most posttranslational modifications, suchas the
initiation of glycosylation, disulfidebond formation and protein
folding occur inthe ER. Transport to the next compartment,the cis
Golgi, occurs by vesicular transport,which is also the way proteins
are transportedthrough the Golgi (intra-Golgi) to the transGolgi
network (TGN) (Rexach and Sheckman,1991). Transport, docking, and
fusion of trans-port vesicles are regulated by proteins gener-ally
refered to as SNAREs (reviewed by Gerst,1999). The anterograde
and/or retrogrademembrane traffic between the ER and Golgioccurs in
vesicles coated with COPI and COPIIproteins (Bednarek et al., 1995;
Schekman andMellman, 1997; Orci et al., 1997). In the TGNproteins
are sorted into distinct vesicles, which
Figure 2. A schematic presentation of the secre-tory route of
yeast S. cerevisiae. The nascentsecretory proteins are translocated
into the lumenof the endoplasmic reticulum (ER), from wherethey are
transported by carrier vesicles via theGolgi to the vacuole, the
plasma membrane, thecell wall or to the growth medium.
Reproductionfrom Saris (1998) with permission.
-
Introduction
9
are destined to the plasma membrane or tovacuoles (Griffiths and
Simons, 1986;Conibear and Stevens, 2000). The sorting tovacuoles
occurs via a late endosome-likeprevacuolar compartment (Horazdovsky
et al.,1995; Gerrard et al., 2000; Götte and Lazar,1999).
The ER signal sequence consists of a shorthydrophobic peptide
with hydrophilic residuesat both ends (von Heijne, 1985; Rapoport
etal., 1996). Polypeptides are translocated intothe lumen of the ER
usually cotranslationally,although some proteins translocate
post-translationally. During co-translational trans-location, the
signal sequence of a nascentpolypeptide is first recognized by the
signalrecognition particle (SRP) (Walter andJohnson, 1994). After
binding to the SRP,translation is arrested until the complex
isbound to the ER membrane via the SRPreceptor. The translocation
of proteins occursthrough translocation channels
containinghetero-trimeric complexes of the Sec61 protein(Hanein et
al.,1996; Hamman et al., 1997).The signal peptides of proteins
which trans-locate posttranslationally are less hydrophobicand are
thus independent of SRP (Ng et al.,1996; Rapoport et al., 1996).
Posttranslationaltranslocation requires a complex of Sec62,Sec63,
Sec71 and Sec72 proteins in additionto the Sec61p translocon
channel (Panzner etal., 1995; Brodsky and Scheckman, 1993). TheBiP
chaperone has a critical role in trans-location. It binds to the
translocating poly-peptide, acting as a molecular ratchet
thuspreventing the backsliding of the protein(Matlack et al.,
1999).
The ER is the main location where secre-tory proteins fold and
it contains severalfolding enzymes and chaperone proteinsessential
for proper protein folding and trans-port (Hong, 1996). The lumen
of the ER is anoxidizing environment and contains highlevels of
calcium, which provides properconditions for chaperones and
disulfide bondformation (Lodish et al., 1992; Hwang et al.,1992).
Signal peptidase and glycosyl-
transferase complexes are functionally asso-ciated, and signal
sequence cleavage andglycosylation, together with protein folding
oc-cur cotranslationally in the ER. In yeast cells,proteins are
glycosylated with N-linked andO-linked glycans. Glycosylation is
assumedto promote correct folding, protect againstproteolysis and
thermal denaturation, as wellas to regulate intracellular
trafficking (Lis andSharon, 1993). In O-linked oligosaccharides,the
first mannose residue is attached to a serineor threonine in the
ER, which is unlike mam-malian cells where mucin-type
O-glyco-sylation is initiated in the Golgi by adding
anN-acetyl-galactosamine to the fully foldedprotein (Tanner and
Lehle, 1987; Lussier etal., 1995; Van den Steen et al., 1998).
O-linkedoligosaccharide chains in yeast can be elon-gated up to 5
mannoses in the Golgi (Fig. 3A)whereas O-glycans in mammalian cells
areelongated by adding mannose, galactose, N-acetyl glucosamine,
fucose, sialylate residuesand/or polylactosamine-extensions
(Varki,1998, Van den Steen et al., 1998). N-glyco-sylation of
secreted glycoproteins in yeast isinitiated in the ER (Helenius,
1994, Heleniuset al., 1992). In yeast, the core oligosaccha-ride
consisting of two N-acetyl glucosamine,nine mannose residues and
three glucose resi-dues is similar to that of higher eukaryotes
(Fig.3B) (Kukuruzinska et al., 1987). Unlikemammalian cells, the
mannose residues are notreplaced by other sugars (N-acetyl
glu-cosamine, galactose, fucose and sialic acid)in the Golgi, but
the outer chains are elongatedby mannose residues only (Fig. 3C)
(Byrd etal., 1982; Tanner and Lehle, 1987).
1.2.4. Optimizing expression
The genetic background of both natural andrecombinant yeast
strains have been found toaffect the quantity and structure of
heterolo-gous proteins (Eckart and Bussineau, 1996).Because
different genetic backgrounds caninfluence transcription and
translation efficien-cies, the secretory pathway, protein
quality,plasmid stability and plasmid copy number, it
-
Introduction
10
may be necessary to screen a number ofdifferent host strains
with varying geneticbackgrounds when optimizing heterologousprotein
expression in yeast (Schultz et al.,1994). Some genetic background
consider-ations that may aid in foreign protein produc-tion in
yeast include: the use of protease-deficient strains, which can
decrease heterolo-gous protein degradation thus increasing
yields(Van den Hazel et al., 1996), and the use ofstrains lacking
hyperglycosylation of N-linked
sites (Schultz et al., 1994; Nakanishi-Shindoet al., 1993).
However, these strains usuallygrow slower than normal strains, but
there arealso yeast strains that require relatively fewgenerations
to obtain high production yields(Bussineau and Shuster, 1994). An
N-termi-nus sequence of human interleukin 1ß couldbe utilized as an
enhancer for heterologousprotein expression in the same way as has
beenreported for two human growth hormones (Leeet al., 1999). And,
as in E. coli, the co-expression of chaperones have been reportedto
assist in the proper folding and secretion offoreign proteins in S.
cerevisiae (Langer et al.,1992; Chen et al., 1994; Robinson et al.,
1995).Furthermore, the overexpression of disulfideisomerase (PDI)
resulted in a 10-fold increasein secreted human platelet-derived
growthfactor, a four fold increase in acid phosphatase(Robinson et
al., 1994), a two to eight foldincrease for five single chain
antibody frag-ments (Shusta et al., 1998), and a 15 to 24-fold
increase in antistasin (Schultz et al., 1994).
1.3. INSECT CELLS (BACULOVIRUS SYSTEM)The baculovirus expression
system (BVS)
is commonly used to express heterologousproteins in insect cells
(Kost and Condreay,1999). Since insect cells are
eukaryotic,proteins expressed in them will be post-translationally
modified in a manner similarto that of mammalian cells (Miller,
1988).Insect cells can be grown as suspension cul-tures, which
permits the use of large-scale bio-reactors for easier production
scale-up. Fur-thermore, unlike mammalian cell lines insectscells do
not require CO2-incubators. The mostcommonly used baculovirus
system utilizedAutographa californica Multiple NuclearPolyhedrosis
virus (AcMNPV) (Jones andMorikawa, 1996) in a cell line derived
fromLepidopteran Spodoptera frugiperda ovariancells (Sf9). The
AcMNPV’s genome is adouble-stranded, circular DNA, approximately130
kilobases in length, and has been fullysequenced (Ayres et al.,
1994). The produc-
Figure 3. Carbohydrate structures of S. cerevisiaeglycoproteins.
(A) The first mannose of an O-linked oligosaccharide is attached to
a serine (Ser)or threonine (Thr) residue in the ER and the chainis
elongated up to pentamannosides in the Golgicompartment. (B) An
N-acetyl glucosamine isattached to an asparagine (Asp) residue to
fromthe core structure of N-linked oligosaccharide inthe ER and (C)
the outer chains are elongated inthe Golgi. The length of the outer
chain variesfrom 2 to 15 mannose residues.
-
Introduction
11
tion systems are commonly regulated by strongpromoters like
polyhedrin and p10 but alsoimmediate early promoters (for example
ie1)have been utilized (Jarvis et al., 1990). Latterexpression
systems gave a continuous andstable expression of human
glycoprotein inboth infected and transformed Lepidopterancells
(Jarvis et al., 1996).
As an alternative to baculovirus expressionsystems, an
expression cassette for continu-ous protein expression by
transformed insectcells have also been developed (Farrell et
al.,1998). The system utilizes the promoter of thesilkmoth
cytoplasmic actin gene, the ie1transactivator gene and the HR3
enhancer re-gion of Bombyx mori MNPV to stimulate geneexpression.
Levels of produced proteins in thissystem are comparable to the
ones producedin BVS (Lu et al., 1997; Keith et al., 1999).
1.3.1. Systems utilizing polyhedrin andp10
In general, wild type baculoviruses exhibitlytic and occluded
life cycles, producing ex-tra-cellular and occlusion derived
virus(ODV), respectively. ODV is embedded inproteinaceous viral
occlusions called poly-hedra (Rohrmann, 1986). Polyhedrin, a 29
kDprotein, is the major component of polyhedra,which in turn are
crystalline occlusion bodiesvisible in the nuclei of infected cells
by lightmicroscopy. The expression of viral genes isregulated in a
successive cascade consistingof 4 distinguishable phases: early,
delayedearly, late and very late (Summers and Smith,1988). During
the very late phase of viralreplication two very abundant mRNAs are
pro-duced, one of which codes for the polyhedrinprotein and other
codes for the p10 protein (10kDa). The p10 polypeptide is involved
in theformation of fibrillar structures found both inthe nucleus
and in the cytoplasm of baculo-virus infected cells. The role of
polyhedra isto protect occluded baculoviruses after thedeath of
infected insects, and its presence isessential for the maintenance
of the virus innature. However, viral propagation in cell
cultures is based on virions which bud frominfected cells, not
on ODV. Thus, baculovirusexpression vectors have been engineered
inwhich the polyhedrin and p10 coding regionsare deleted, but their
strong promoters are re-tained to express foreign proteins in
largeamounts in the late and very late phases ofinfection (Smith et
al., 1983b; Kost andCondreay, 1999).
In the baculovirus expression system utiliz-ing the p10
promoter, the sequence encodingthe p10 protein as well as the
entire polyhedrinlocus, including its promoter, have beendeleted
(Vlak et al., 1990; Roelvink et al.,1992; Bonning et al., 1994;
Naggie andBentley, 1998). The remaining p10 promoteris used to
express recombinant protein se-quences. In this way, expression of
both thenative polyhedrin and p10 proteins are abol-ished, which
reduces background problemscaused by the expression of large
amounts ofvery late native proteins while increasing theexpression
of foreign recombinant proteins.Furthermore, the p10 promoter is
activatedearlier in the infection cycle than the poly-hedrin
promoter (Roelvink et al., 1992;Bonning et al., 1994). The absence
of the p10protein results in delayed cell lysis, and therebyan
extended period of recombinant protein syn-thesis. The yields of
two reporter enzymes, ju-venile hormone esterase and
ß-galactosidasewere higher under the p10 promoter comparedto
polyhedrin controlled expression (Bonninget al., 1994).
1.3.2. Recombination
In baculovirus expression systems the inser-tion of genes coding
for foreign proteins canbe accomplished by homologous
recombina-tion, site-specific transposition (Bac-to-Bac)or
insertion directly into the viral genome invitro. In homologous
recombination, the geneof interest is cloned into a transfer
vectorcontaining a baculovirus promoter flanked bybaculovirus DNA
derived from the polyhedringene. After transfection into insect
cells, thegene is inserted into the genome of the parent
-
Introduction
12
virus by homologous recombination, at a rateof approximately 0.1
to 1% (Smith et al.,1983a). Recombinants are identified by
theiraltered plaque morphology, which appears as‘occlusion minus’
plaques. A higher rate ofrecombination can be achieved when the
par-ent virus genome is linearized at site(s) nearthe target site
of foreign gene insertion (Kitts
et al., 1990). Kitts and Possee (1993) reportedrecombination
rates approaching 100% whenthey used linearized viral DNA that is
miss-ing an essential portion of the baculovirus ge-nome downstream
from the polyhedrin locus.Nevertheless, by this method it can take
morethan a month to purify the plaques, and con-firm the desired
recombinants. A faster methodto generate recombinant baculovirus
can beachieved by utilizing baculovirus Ac-omega,which contains an
unique restriction site down-stream of the polyhedrin promoter
(Ernst etal., 1994). By this method recombinant viruseswere
obtained 8 days posttransfection andaccording to PCR analysis the
non-recombi-nant background was 25 fold lower than thatof the
recombinant viral DNA. Anothermethod utilizing site-specific
transposition isalso relatively fast (7 - 10 days) because
plaquepurification or virus amplification are notneeded (Luckow et
al., 1993). In this method,the foreign gene is cloned into a
baculovirusshuttle vector (bacmid) that can replicate in E.coli,
but can also infect susceptible lepi-dopteran insect cells (Fig.
4). Bacmid is a re-combinant virus that contains a mini-F
repli-con, a kanamycin resistance marker, andattTn7, the target
site for the bacterialtransposon Tn7 (Leusch et al., 1995).
Expres-sion cassettes comprising a baculoviruspromoter driving
expression of a foreign genethat is flanked by the left and right
ends ofTn7, can transpose to the target bacmid in E.coli when Tn7
transposition functions are pro-vided by a helper plasmid.
1.3.3. Optimizing expression
The density at which cells are infected, andthe multiplicity of
infection (m.o.i.) greatlyaffects the expression of heterologous
proteins(Power et al., 1994; Licari and Bailey, 1992;Wong et al.,
1995; Klaassen et al., 1999). Forinstance, when the integral
membrane proteinbovine rhodpsin was expressed in largeamounts, the
highest volumetric yields wereobtained with an m.o.i. of 0.01
during early tomid-exponential growth (Klaassen et al.,
Figure 4. Schematic outline of the generation ofrecombinant
baculovirus with a bac-to-bac expres-sion system. A donor vector
carrying the gene ofinterest flanked by a Tn7-element is
transformedto E. coli. The Tn7 element can integrate into theattTn7
target site in the presence of transpositionproteins provided by a
helper plasmid. Recombi-nant bacmids are selected using their
antibioticresistance and recombinant bacmid DNA is usedto transfect
insect cells.
-
Introduction
13
1999). For easier purification, and cost savingreasons, it is
possible to produce heterologousproteins in Sf9 cells in serum-free
and pro-tein-free media. The cells were graduallyadapted to
serum-free media in monolayercultures and reached a doubling time
ofapproximately 25 h, compared with 18 h inserum containing medium.
Furthermore, thecontrol of proteolysis in insect cells has a
im-portant role when optimizing the yield ofrecombinant protein
(Naggie and Bentley,1998). Like in S. cerevisiae, unwanted man-nose
residues can be added to secreted glyco-proteins and they lack
penultimate galactoseas well as terminal sialic acid residues
(Jarvisand Finn, 1996). The immediate earlypromoters (for example
ie1), have been shownto be beneficial for protein production
e.g.,when highly glycosylated or otherwise modi-fied proteins are
to be produced (Bonning etal., 1994; Chazenbalk and Rapoport,
1995).
1.4. MAMMALIAN EXPRESSION SYSTEMSUTILIZING VACCINIA AND
ALPHAVIRUSVECTORS
In some cases, mammalian cell systems arethe only possible way
to produce properly pro-cessed and active foreign proteins. Gene
trans-fer into mammalian cells may be performedeither by infection
with a virus carrying therecombinant gene of interest (reviewed
byMakrides, 1999) or by direct transfer of plas-mid DNA (Geisse and
Kocher, 1999).
1.4.1. Vaccinia virus vector
Recombinant vaccinia virus vectors havegenerally been
acknowledged as versatile toolsfor the expression of foreign genes
(reviewedby Moss, 1996). Since vaccinia is infectiousto man, safety
aspects must be taken into con-sideration, especially when working
withlarge-scale preparations. To circumvent safetyproblems, an
avian host-restricted vaccinia,modified vaccinia Ankara (MVA), can
be used(Sutter et al., 1994; Wyatt et al., 1995). Fur-thermore, MVA
does not produce the rapid
cytophatic effect accompanied by destructionof the cell
monolayer seen with the standardreplication strains of vaccinia
used. MVA hasbeen shown to efficiently produce foreignproteins in
several mammalian cell lines usingeither the homologous vaccinia
promoter, p11(Sutter and Moss, 1992) or a hybrid
vaccinia/bacteriophage T7 promoter system (Wyatt etal., 1995). In
the vaccinia/T7 polymerase hy-brid system, the vaccinia virus
contains the T7RNA polymerase gene, which is under thecontrol of an
early vaccinia promoter. Theforeign gene to be expressed is cloned
into aseparate expression vector under the controlof the T7
promoter. After the host cells areinfected with the vaccinia/T7
virus, the cellsare transfected with the expression
plasmid.Problems with vaccinia systems include theircytopathic
nature, and dependence on efficienttransfection rates. Mammalian
cell lines canbe engineered so that the genes to be trans-fected
are stably integrated into the cell´s ge-nome, but then a tightly
controlled-induciblepromoter must be used. This method is
espe-cially advantageous when foreign protein pro-duction must be
scaled-up. Furthermore, ho-mologous vaccinia promoter systems can
bemore effective in producing secreted foreignproteins as compared
to the heterologous T7promoter system (Pfleiderer et al.,
1995).Another vaccinia virus based expression sys-tem utilizes
defective vaccinia virus lackingthe D4R open reading frame, and a
comple-mentary cell line providing the D4R gene prod-uct (Himly et
al., 1998). Experiments donewith human secreted proteins (i.e.,
factors VIIand XI) showed that the defective systemproduces more
secreted proteins than the wildtype vaccinia. Surprisingly,
recombinanthuman factor VII was more efficientlyproduced using the
defective vaccinia recom-binant under non-complementing
conditions(Himly et al., 1998). This suggests that thepersistence
of early phase vaccinia replication,combined with a delay in the
shutoff of hostprotein synthesis, can be advantageous forforeign
protein production.
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Introduction
14
1.4.2. Alphavirus vectors
Semliki Forest (SFV) and Sindbis (SIN)viruses together with
Venezuelan equineencephalitis virus are used as vectors for
theexpression of heterologous proteins in manydiverse cells types
(Makrides, 1999; Garoffand Li, 1998; Agapov et al., 1998).
Alphavirusreplicon vectors are self-replicating RNAmolecules which
include the genes for non-structural proteins (Nsps), while lacking
thegenes of the viral structural proteins. Insteadof the structural
genes, cloning sites have beenengineered to accept foreign genes
for expres-sion. The replicons are introduced into hostcells by
transfection either as RNA, synthe-sized in vitro, or as DNA under
the control ofan eukaryotic promoter. Alternatively, the
re-combinant alphavirus RNA can be packagedinto viral particles in
cells that have beencotransfected with replication competent,
butpackaging incompetent helper RNA coding forthe alphavirus
structural proteins (Bredenbeeket al., 1993). Although,
recombinationbetween vector and helper genomes can gen-erate a
fully infectious virus, this usuallyhappens at very low frequencies
(Berglund etal., 1993). One way to prevent this recombi-nation is
to use conditional mutations to limitthe infectivity of any such
recombinants(Smerdou and Liljeström, 1999). In an im-proved
packaging system, the helper virus hasbeen modified so that the
structural proteinsare expressed in two separate plasmids.
As in vaccinia systems, alphavirus infectionsshuts off the
host’s own protein synthesis,which makes protein purification
easier.Compared to vaccinia, SFV is less cytopathicto the host, and
thereby potentially more effi-cient in foreign protein production
(Liljeströmand Garoff, 1991). Furthermore, non-patho-genic
alphavirus vectors have been developed.For example, a Sindbis virus
derivative, whichhas low host cell pathogenicity due to a
singlepoint mutation in Nsp2, demonstrates apersistent infection,
while its replication rateis as high as the one of the wild type
SIN(Dryga et al., 1997). Although expression of
foreign proteins using alphaviruses derivedvectors can generate
very high yields (up to25% of total cellular proteins), cloning of
largeforeign genes into replicons can be problem-atic (Liljeström
and Garoff, 1991). Foreignsequences of about 2 kb are usually
stable inSIN vectors, whereas various inserts over 3kb were rapidly
deleted suggesting that thepackaging capacity of the virion had
beenexceeded (Pugachev et al., 1995). Besidestissue and suspension
culture applications ofalphavirus replicons, in situ infections
andforeign protein expression have been accom-plished in the
neurons of rat hippocampalslices (Schlesinger and Dubensky, 1999).
Thissystem has been used to produce ß-galactosi-dase, tissue
plasminogen, chloramphenicolacetyl transferase, hemagglutinin
andHepadnavirus proteins. Furthermore, deriva-tives of alphavirus
vectors have been used todirect foreign protein expression in
specificcell types in a tissue (Huang, 1996; Ohno etal., 1997).
1.4.2.1. Semliki Forest virus
Semliki forest virus (SFV) is an envelopedRNA virus with a
single strand genome ofpositive polarity. SFV belongs to the
Alpha-virus genus which in turn is a member of thefamily
Togaviridae. The prototype virus ofSFV, used in laboratories, is
considered to benonpathogenic to humans and has been longstudied as
a model virus for the Alphavirusgenus. SFV enters the cell via
receptor medi-ated endocytosis, resulting in nucleocapsidliberation
of its RNA genome into the cyto-plasm (Helenius et al., 1980).
Two-thirds ofthe genomic 42S RNA (total length ca. 11.5kb) is
translated into a polyprotein, which isthen autoproteolytically
cleaved into four non-structural proteins: Nsp1 - Nsp4 (Fig. 5)
(re-viewed in Kääriäinen et al., 1987; Strauss andStrauss, 1994).
In the initial stages of SFVinfection, a RNA replication complex,
isformed from the intermediate translation prod-ucts Nsp123 and
Nsp4 and is needed torecognize the 42S RNA plus strand for
tran-
-
Introduction
15
scription of the negative strand 42S RNA(Lemm and Rice, 1993a,
1993b). Later ininfection the intermediate Nsp123 is
pro-teolytically processed into the final products,Nsp1, Nsp2 and
Nsp3, after which the tran-scription of the minus strand 42S RNA
willbe terminated. According to the geneticcriteria, all
nonstructural proteins are neededto form the replication complex.
Replicationoccurs in association with modified endo-somes and
lysosomes called cytopathic vacu-oles type I (CPVI) (Froshauer et
al., 1998;Peränen and Kääriäinen, 1991). The structuralproteins:
capsid protein and envelope glyco-proteins E1, E2 and E3 are
transcribed from asubgenomic 26S RNA (reviewed in Straussand
Strauss, 1994). The functions of individualNsps are briefly
reviewed below.
1.4.2.1.1. Nsp1Nsp1 (537 aa) is multifunctional enzyme
catalyzing the capping of viral mRNA. Firstly,it is a
methyltransferase (MT) (Mi et al., 1989,Mi and Stollar, 1991;
Laakkonen et al., 1994).Nsp1, like the methyltransferase purified
fromthe vaccinia virion can methylate GTP in thepresence of
S-adenosyl-L-methionine(AdoMet) (Laakkonen et al., 1994, Martin
andMoss, 1976). Secondly, Nsp1 has been shown
to form a covalent complex with 7-methyl-GMP thus providing at
least the first reactioncatalyzed by guanylyltransferases (GT)
(Aholaand Kääriäinen, 1995). The capping reactionof viral mRNA
catalyzed by Nsp1 differs fromthe capping of eukaryotic mRNA and
manyother viral mRNA by requiring methylationof guanine before
covalent complex formationbetween the guanylyltransferase and
7-methyl-guanylate (Fig. 6) (Ahola and Kääriäinen,1995). The
7-methyl-guanylate is probablybound to Nsp1 through a phosphoamino
bondeither on a lysine or on a histidine. Further-more, Nsp1 also
assists in the initiation of mi-nus strand synthesis (Hahn et al,
1989b).
When Nsp1 is expressed alone in HeLa cellsby transfection, it
associates with the plasmamembrane as well as with endosomes and
ly-sosomes (Peränen et al., 1995). The tight mem-brane association
of Nsp1 is partly due topalmitoylation on amino acids
Cys418-Cys420
(Peränen et al., 1995; Laakkonen et al., 1996).When the
palmitoylation sites are mutated, theprotein remains enzymatically
active andbound to membranes (Laakkonen et al., 1996),though less
tightly as compared to the wildtype (i.e., the nonpalmitoylated
mutant formcould be released from the membranes with 1M NaCl,
whereas wt Nsp1 could be releasedonly with 50 mM sodium carbonate,
pH 12).
Figure 5. Proteolytic processing of the nonstructural and
structural proteins of Semliki Forest virus.
-
Introduction
16
In addition, the morphology of cells infectedor transfected with
nonpalmitoylated Nsp1differs from wt by showing fewer filopodialike
structures than the wt Nsp1.
1.4.2.1.2. Nsp2According to what is presently known, Nsp2
(799 aa) is responsible for three differentenzymatic activities
in SFV replication. Firstly,it is an autoproteinase containing a
papin-likethiol proteinase at its C-terminal region (Hardyand
Strauss, 1989). Secondly, it is an RNAhelicase (Gomez de Cedron et
al., 1999),which has single stranded RNA-stimulatedATPase and
GTPase activities (Rikkonen etal., 1994). Thirdly, Nsp2 is a
triphosphatase,which catalyzes the first reaction in mRNAcapping
(Vasiljeva et al., 2000). Moreover,Nsp2 has been reported to act on
the regula-tion of negative strand synthesis, and isrequired for
the synthesis of the subgenomic26S RNA (Hahn et al., 1989b). About
half ofthe Nsp2 is transported into the nucleus duringinfection,
and this transport is mediated bynuclear signal sequences (Peränen
et al., 1990;Rikkonen et al., 1992). SFV in which thenuclear
targeting signal of Nsp2 is removed,has been reported to be
apathogenic in mice(Rikkonen et al., 1996).
1.4.2.1.3. Nsp3Nsp3 (482 aa) is an essential subunit for the
replication complex although its specific func-tions are still
not known. Studies of SIN Nsp3have suggested that the polyprotein
interme-diates have distinct essential functions in thesynthesis of
negative strands during the earlyphases of RNA replication (Lemm et
al., 1994;Shirako and Strauss 1994). SIN Nsp3 has beenreported to
affect negative-strand RNA syn-thesis, and possibly also the
synthesis ofsubgenomic mRNA (Wang et al., 1994;LaStarza et al.,
1994b). When the localizationof SFV Nsp3 was studied by indirect
immun-ofluorescence it was found to be mainly invacuole like
structures both in infected as wellas transfected cells (Peränen
and Kääriäinen,1991).
The N-terminal region of different Nsp3s arevery conserved among
the alphaviruses, andpart of that sequence has homology to
rubellavirus, hepatitis E virus, and coronavirusessequences
(X-motif, Koonin and Dolja, 1993).Recently, through genome
sequencing, it hadbecome apparent that this domain can be foundin
bacteria, animals and plants and it showshomology to the nonhistone
region of macrohistone 2 (Pehrson and Fuji, 1998). TheNational
Center for Biotechnology Informa-tion (NCBI) has assigned this
domain as
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Figure 6. Reactions involved in capping of alphavirus mRNA (A)
and eukaryotic mRNA (B).
-
Introduction
17
DUF27 with an unknown function. Moreover,this domain has
significant homologue to oneyeast ORF which has been associated
withadenosine-diphosphate-ribose-1-phosphate(Appr-1"-p) processing
activity (Martzen etal., 1999). Conservation of this region
duringevolution suggests an important function(s) forthis
domain.
The C-terminal part of alphavirus Nsp3 isnot conserved among
alphaviruses, and var-ies both in sequence and in length from 134aa
(Middel-burg virus) to 246 aa (O’nyong-nyong virus) (Strauss and
Strauss, 1994). Thisdomain is rich in acidic residues, as well as
inserine and threonine and appears to be devoidof any predicted
secondary structures. Theregion also contains duplicate amino acid
(aa)sequences i.e., ADVPEPA, PAPR andTFGDFD, suggesting that this
region hasevolved through duplication events. Nsp3 isthe only
alphavirus nonstructural protein modi-fied by phosphorylation
(Peränen et al., 1988;Li et al., 1990).
1.4.2.1.4. Nsp4Even though direct evidence is still lacking,
Nsp4 (614 aa) most probably is the catalyticsubunit of the
alphavirus RNA-replicationcomplex (Barton et al., 1988). Nsp4
hasconserved a GDD motif found in other viralRNA polymerases
(Argos, 1988), and a SINtemperature sensitive mutant, ts6, has
beenreported to fail in the synthesis of RNA due toa mutation in
Nsp4 (Sawicki et al., 1981a1981b, Hahn et al., 1989). Nsp4 has also
beenreported to be an autoproteinase (Takkinen etal., 1990).
2. PURIFICATION STRATEGIES FORRECOMBINANT PROTEINS
Purification is an important step in the pro-duction of
recombinant proteins. The char-acteristics of industrial-scale
purificationschemes, such as conventional chromatogra-phy, have a
significant impact on the finalcost of production. It is often more
efficient
to use one of the available tags to ‘fish out’the target
protein. The purpose for which theprotein will be used determines
requireddegree for its purity and authenticity. Topurify
intracellularly produced proteins thecells are harvested and lysed,
which naturallycontributes to the complexity of the proteinmixture.
The advantage of intracellular ver-sus secreted protein is the
volume to behandled, i.e., the secreted proteins usuallyexist
diluted in the culture medium. Isolationof a desired protein from
the medium followsthe general scheme: (1) concentration by
pre-cipitation, ultrafiltration, batch adsorption orpartition in an
aqueous phase system (2)enrichment by chromatography or
partition(3) high resolution purification: by chroma-tography
and/or immuno adsorption (4) finalconcentration (Menge et al.,
1987). If fetalcalf serum (FCS) is used in cultivation,bovine serum
albumin and globulins will bethe most abundant proteins in the
superna-tant. The interaction of albumin especiallywith hydrophobic
proteins represents asignificant problem for the effective
purifi-cation of minor constituents.
Even though there are many existingexamples as well as
heuristics and complexalgorithms for suggesting the potential
ofpurification processes, no universal schemehas been developed
which could be appliedto all proteins. There are a variety of
differ-ent methods which can be used to purify pro-teins, but only
chromatographic purificationmethods such as affinity, ion
exchange,reversed phase and gel filtration chromatog-raphy will be
briefly reviewed here.
2.1. CONVENTIONAL CHROMATOGRAPHYChromatographic methods (size
exclusion,
ion exchange, reversed phase, hydrophobicinteraction and
affinity based) can be utilizedeither in traditional, low pressure
or high-performance liquid chromatography instru-mentation. In
addition to liquid-solid chroma-tography there is liquid-liquid
chromatogra-
-
Introduction
18
phy which is analogous to gas-liquid chroma-tography (Cichna et
al., 1995). There are alsomany other types of chromatography
methodse.g., liquid adsorption chromatography,
frontalchromatography, displacement chromatogra-phy covalent
chromatography and membranechromatography (Mao et al., 1993; Heeter
andLiapis, 1998; Freitag, 1999; Caldas et al.,1998; Zeng and
Ruckenstein, 1999).
2.1.1. Ion exchange chromatography
Ion exchange chromatography (IEC) hasbeen the most widely used
technique for theisolation and purification of biological
macro-molecules since the 1950s (Choudhary andHorváth, 1996). IEC
is able to separate almostany type of charged molecules, from large
pro-teins to small nucleotides and amino acids. Theion exchanger
are insoluble solid matricescontaining fixed ionogenic groups which
bindreversibly to sample molecules (proteins, etc.).Desorption is
then brought about by increas-ing the salt concentration or by
altering thepH of the mobile phase. The two major classesof ion
exhangers are cation exchangers andanion exchangers, having
negatively and posi-tively charged functional groups,
respectively.Ion exchange containing diethyl aminoethyl(DEAE) or
carboxymethyl (CM) groups aremost frequently used.
Since the protein is covered by a hydrophiliclayer,
electrostatic interactions have a majorrole in the retention of
proteins. In addition tosize, geometric form as well as
hydrophopicand van der Waals interactions affect the sepa-ration
(Stålberg, 1999). Nevertheless, the ma-jor property which govern
the adsorption toan ion exchanger is the net surface charge ofthe
protein. Since surface charge is the resultof weak acidic and basic
groups on proteins,separation is highly pH-dependent. The opti-mum
pH range for IEC for many proteins iswithin 1 pH units of the
isoelectric point. Manyretention models have been examined but
thecomplexity of the adsorption process for a pro-tein to solid
surfaces makes it difficult to con-struct physical models for the
interactions
(Stålberg, 1999). Gradient elution with increas-ing salt
concentration is most commonly usedin the IEC. The higher the net
charge of theprotein, the higher the ionic strength neededto bring
about desorption. Thus, to optimizeselectivity in ion exchange
chromatography,the pH of the running buffer is chosen so
thatsufficiently large net charge differences amongthe sample
components are created.
2.1.2. Reversed phase chromatography
Reversed phase chromatography (RPC) canbe utilized to separate
compounds accordingto their hydrophobicity (Geng and Regnier,1984).
Unlike many other methods RPC isable to separate closely related
and structur-ally disparate substances, even at picomolarlevels
(Aguilar and Hearn, 1996; Pearson etal., 1982). In RPC, silica
particles coveredwith chemically-bonded hydrocarbon chainsrepresent
the lipophilic phase (C2 to C18),while an aqueous mixture of an
organic sol-vent surrounding the particle represents the
hy-drophilic phase (Henry, 1991; Zhou et al.,1991). Depending on
the extractive power ofthe eluent, a greater or lesser part of the
samplecomponent will be retained reversibly by thelipid layer of
the particles. The partitioning ofthe sample components between the
twophases will depend on their respective solu-bility
characteristics. Less hydrophobic com-ponents will end up primarily
in the hydro-philic phase while more hydrophobic ones willbe found
in the lipophilic phase. This can beaffected by the addition of an
organic solventwhich is soluble in the hydrophilic phase.Some
commonly used organic solvents, inorder of increasing
hydrophobicity are metha-nol, propanol, acetonitrile, and
tetrahydrofu-ran. Separated components can be directlysubjected to
further analysis such as Edmansequencing or electrospray mass
spectrometry.
The ability of a stationary phase (lipophilicphase) to
discriminate between two compo-nents is reflected by the volume
between thepeak maxima of the corresponding zones afterpassing
through the column (Aguilar and
-
Introduction
19
Hearn, 1996). Along with partitioning, mecha-nism adsorption
operates at the interfacebetween the mobile and the stationary
phases(Melander et al., 1984). Thus, the retention ofhydrophobic
components will be greatly in-fluenced by the thickness of the
lipid layer. AC18 layer is able to accommodate more hydro-phobic
material than C8 or C2 ones. For hy-drophilic components, changing
from a C18to a C2 layer influences the separation verylittle since
only the surface area of the lipidlayer is active. The mobile phase
can be con-sidered as an aqueous solution of an organicsolvent, the
type and concentration of whichdetermines the extractive power.
Moreover,according to experimental data, componentsinteract with
the chromatographic surface inan orientation-specific manner (Chicz
andRegnier, 1990; Regnier, 1987). RPC has beenutilized for
purification of a variety of proteinsand peptides e.g., aprotinin,
cytochrome C,bovine serum albumin, fibrinogen, insulin andlysosyme
(Honda et al., 1992; Nimura et al.,1992). Even though some proteins
have beenreported to maintain their native structureduring RPC
(e.g., insulin, thyroid-stimulatinghormone, growth hormone), in
most casesdenaturing conditions are required whichmight limit the
use of RPC (Welinder et al.,1986; Welinder et al., 1987; Forage,
1986;Chlenov et al., 1993).
2.1.3. Gel permeation chromatography
The principle of gel permeation chromatog-raphy (GPC; size
exclusion or gel filtrationchromatography) is based on
molecularvolumes. Large molecules are excluded fromthe matrix,
whereas intermediate size moleculecan partly enter and only small
molecules canfreely enter the matrix. The porous threedimensional
matrix acts as a steric barrier tosolute molecules as they attempt
to equilibratewith liquid inside and outside the bead.
Whilepurifying the protein GPC can also be used toestimate
approximate molecular weights.Furthermore, gel filtration can be
used fordesalting or buffer changing when the
fractionated size of the gel is small (e.g., Bio-Gel 6, Sephadex
G-25) allowing the proteinto elute in the void volume.
The choice of the appropriate column typedepends on the
molecular size and physicalproperties of the proteins to be
separated. Afraction of the internal volume which is ac-cessible to
a solute molecule can be describedby a constant Kd, which can be
calculated withexperimentally determined elution volumes.A column
should be chosen so that separa-tions occur in the linear part of
the Kd vs. themolecular weight plot (van Dijk and Smit,2000).
However, it is practically impossibleto adjust the pH and ionic
strength so that theproteins are in an equal state, since at the
iso-electric point the shape of globular and poly-mer coil proteins
relieve compact and softsphere, respectively. Furthermore, the
ionicstrength of the buffer used may affect the sizeof the
proteins. Experiments with neutral dex-trans and charged proteins
showed that theeffective protein size increases with decreas-ing
ionic strength due to the reduction in elec-trostatic shielding
(Pujar and Zydney, 1998).Thus, although GPC is viewed as
size-basedseparation process, there is considerable evi-dence for
the importance of electrostatic in-teractions as well. GPC has been
used in therenaturation procedure (Müller and Rinas,1999; Batas and
Chaudhuri, 1999). For ex-ample heterodimeric platelet-derived
growthfactor was purified from inclusion bodies af-ter denaturation
with Gdn-HCl (Müller andRinas, 1999). Renaturation of this growth
fac-tor involved folding into an active hetero-dimeric form during
GPC while circumvent-ing aggregation observed when refolding
wascarried out by dilution.
2.2. AFFINITY CHROMATOGRAPHY
Affinity chromatography together with re-combinant
DNA-technology offers a simpleand fast technique to purify proteins
to highpurity with a single purification step (Scouten,1991).
Fusion can be made on either side or
-
Introduction
20
both sides of the target gene depending on spe-cific
application, but the majority of fusionproteins place the tag at
the N-terminus of theprotein (Nilsson et al., 1997). Genetic
manipu-lation of the protein can be used to form acleavage site,
which helps to remove theaffinity tag after purification thereby
resultingin an intact protein. Although affinity chro-matography
can be used for laboratory-scalepurification, its utilization on a
preparativescale can represent a major cost for the finalprotein
product.
Successful separation by affinity chromatog-raphy requires that
a biospecific ligand is avail-able, and that it is covalently
attached to a chro-matographic bed material. It is important
thatthe biospecific ligand (antibody, enzyme, orreceptor protein)
retains its specific bindingaffinity for the substance of interest
(antigen,substrate, or hormone). These interactionstypically have
high affinities (Kd < 10-6 M),yet are reversible when conditions
are changed(Wilchek et al., 1984). Due to the specificityof this
recognition, it is often possible to ob-tain 100-, 1.000- or even
100.000-fold in-creases in purity of a protein sample (Clausenet
al., 1990). The packing material used, calledthe affinity matrix,
must be inert and easilymodified. Agarose is the most common
sub-stance used as a matrix, in spite of its relativehigh costs.
The ligands, or “affinity tails”, thatare inserted into the matrix
can be geneticallyengineered to possess a specific affinity. In
aprocess similar to ion exchange chromatogra-phy, the desired
molecules adsorb to theligands on the matrix until desorption is
car-ried out e.g., with a high salt concentration, acompetition
reaction (e.g., imidazole), strongchelating agents and/or low pH.
Fouling of thematrix can occur when a large number of im-purities
are present, therefore, this type of chro-matography is usually
implemented late in theprocess. In addition to most common
affinitychromatography utilizing HPLC, there are alsoother
techniques which involve affinity, suchas affinity precipitation,
affinity partitioningof proteins using aqueous two-phase
system,
foam fractionation and dye ligand chromatog-raphy (Hoshino et
al.,1998; Birkenmeier et al.,1984; Lockwood et al., 1997; Boyer and
Hsu,1993).
2.2.1. Affinity tags
To date, a large number of different fusionpartners that range
in size from one amino acidto whole proteins capable of selective
interac-tion with ligand immobilized onto a chroma-tography matrix,
have been described (Nilssonet al., 1997). Although a multitude of
systemshave been introduced, no single affinity fu-sion partner is
ideal for all expression or puri-fication systems. Some of the most
commonlyused tags are reviewed below and listed inTable 1.
In 1975 Porath and co-workers introduceda method based on the
interaction between theside chains of certain amino acids,
especiallyhistidines, on a protein surface and immobi-lized
transition metal ions. Immobilized metalaffinity chromatography
(IMAC) systems havethree basic components: an electron donorgroup,
a solid support and a metal ion. Themetal ion (usually Ni2+, Co2+,
Cu2+ or Zn2+) isrestrained in a coordination complex where itstill
retains significant affinity towards mac-romolecules (Porath,
1992). The use ofpolyhistidine tags has been demonstrated ina wide
range of host cells including E. coli, S.cerevisiae, insect cells
as well as in mamma-lian cells (Table 1). IMAC is usually
performedunder nondenaturing conditions, but it is alsocompatible
with non-ionic detergents allow-ing highly denaturing conditions
with urea andguanidium-HCl (Smith and Roth, 1993). Alsoan organic
solvent (isopropanol) can be usedto increase the purification
efficiency (Frankenet al., 2000). The elution is usually
performedby competition with imidazole, lowering pH,or by adding
strong chelating agents. IMACis particularly suitable for
preparative groupfractionation of complex extracts and bio-fluids,
but can also be used in the high-perfor-mance mode. Regardless of
the relativelysmall size of the his-tag, is has been reported
-
Introduction
21
to affect the activity of some finally purifiedrecombinant
proteins (Janssen et al., 1995;Pekrun et al., 1995; Büning et al.,
1996).
Maltose binding protein (MBP) from E.coli is frequently used as
a fusion partner forproteins. MBP (40 kDa) is a periplasmic
pro-tein and thereby can be employed to inducethe secretion into
the periplasm. A mixture ofcellulose and starch can