E. coli is your friend - uni-leipzig.de · separation of cells from supernatant diluted, low protein yield cell disruption isolation renaturation renaturation problems, low ionic

E. coli is your friend

Kathrin EngelMolecular Biochemistry

E. coli

11/29/1857 – 02/15/1911

• procaryots enterobacteriaceae (greek: entero = intestine)

• part of the intestinal flora, symbiosis

• gram negative, rod-shaped, eubacterium

• facultative anaerobic O2 consumption or anaerobic fermentation

• exercises the intestinal immune system production of IgA by host

• produces vitamin K

• facultative pathogen pathogen if extraintestinal

E. coli

indicates water pollution by faecesbiosynthesis of insulin and amino acids because E.coli is resident in the human intestinal flora there are no allergic reactions to E.coli-produced molecules

facultative pathogen

• sepsis• urinary tract infection• meningitis• wound infection• peritonitis• cholecystitis• cholangitis

obligatory pathogen

• diarrhea • enteritis • enterocolitis

Pathogenicity of E. coli

• separation of cells after duplication of a DNA molecule

• generation time at 37°C in complete medium 20 – 30 minin minimal medium 60 – 90 min

• cultivation on agar plates (Robert Koch 1880)

• grows easily in a simple nutrient broth in culture bottles

Reproduction of E. coli

37°C

• LB (Luria-Bertani) medium

• liquid and solid medium must be autoclaved

• antibiotics and amino acids are degraded by autoclaving

• add antibiotics only prior to use of liquid medium

• add antibiotics only below 50°C to solid medium

• plates should not be stored longer than 1 month

Growth and culture of E.coli in the lab

Macromolecules of an E. coli cell

11

80> 1,000

3 x 104

3 x 104

4 x 105

103

RNA16S rRNA23S rRNAtRNAmRNA

12 - 4DNA

> 3,000106proteins

typesnmacromolecule

• 4.65 x 106 bp encode 4,288 protein coding genes

• circular and closed nucleoid

• superhelical structure– IHF binds AT-rich regions and causes bending

– HU protein stabilizes bending and crossing of DNA strains

– DNA Topoisomerases: type II (gyrase) superhelical turns

type I relaxation of superhelical turns for replication or transcription

The genome of E. coli

gyrase inhibitors = antibiotics; inhibition of gyrase leads to an extension of the nucleoid DNA loses its compact structure and expands bacterial cell bursts

origin

terminus

• naturally in bacteria (0 to 20 copies)

• circular dsDNA molecules

• separately from the main bacterial chromosome

• replicate independently from the bacterial chromosome

• horizontal gene transfer within a population of microbes

• selective advantage:– ampicillin resistance

– cloacin production

– multi drug resistance

– enterotoxin production

Plasmids in nature

• F plasmids– carry information for own transfer from cell to cell (conjugation)

• R plasmids– encode resistance to antibiotics

• degradative plasmids– carry specific sets for degradation of unusual chemicals

• cryptic plasmids– carry no functional coding gene

Types of plasmids

Exchange of genes

• donor contains F plasmid (fertility)• F plasmid can be isolated from the chromosome (F+ cells)

integrated into chromosome (hfr cells)

bacterial chromosome

F+ (donor) F- (recipient) conjugation tubereplication of

F factor

two F+ cells

Characteristics of a plasmid

circular DNA

cloning site

antibiotic resistance gene

origin of replication

name

• different numbers of plasmid molecules in one cell

• pBR322 20 – 30 copies

• pUC 200 – 300 copies

• low copy plasmids can get lost

High- and low-copy plasmids

pBR322

Prominent expression vectors

cloning site

• mammalian expression vector:– used for amplification of plasmid

– isolation of plasmid-DNA

– transfection of mammalian cells portein production in mammalian cells

– promotor upstream the target gene is a mammalian promotor

– protein produced = ß lactamase

• bacterial expression vectors:– used for expression of proteins in bacteria

– bacterial promotor upstream the target gene

bacterial and mammalian expression vectors

• target genes expressed under T7 promotor

• exclusively for expression of cloned genes

• high expression levels of genes that are not expressed efficiently in other systems

• not recognized by E.coli RNA polymerase

• genes are “off” in non-expression hosts no plasmid instability

• transfer of plasmids into expression hosts containing chromosomal copy of T7 pol

• expression induced by IPTG or lactose

• 2 components: bacteriophage T7 RNA polymerase, plasmid vector with bacteriophage T7 promotor upstream the gene to be expressed

The pET system

• isopropyl-ß-D-thiogalactopyranosid

• inducer of the lac operon

• binds lac repressor

• is not metabolized by E.coli

• used to produce recombinant proteins by expression of cloned genes

• cloned gene under lac promotor

IPTG

plasmids as tools for gene cloning

INSERT

VECTOR PLASMID

LIGATION

TRANSFORMATION IN E.COLI

Transformation of bacterial cells

CaCl2

bacterial cell

competentbacterial cell

42°C(30‘‘-1‘)

transformedbacterial cell

• uptake of DNA into a cell• chemically induced competence

• transformation of competent cells with 1 ng control plasmid containing an antibiotic resistance gene

• plate onto LB-agar plates containing relevant antibiotics

• compare number of colonies obtained with control plasmids with those of interest

• transformation of cells with 20 µl buffer or water

• plate on LB-agar plate containing relevant antibiotics

• absence of colonies indicates an active antibiotic

Transformation efficiency and antibiotic activity

• untransformed cells no growth on medium with antibiotics

• E.coli with plasmid growth

Selection I

• untransformed cells no growth on medium with antibiotics

• E.coli with plasmid growth

• untransformed cells no growth on medium with antibiotics

• E.coli with plasmid growth

Selectable markers

• agar plate contains antibiotic and X-gal

• LacZ gene on the plasmid encodes ß-galactosidase

• ß-galactosidase metabolizes X-gal

• colonies containing antibiotic resistance gene grow

• w/o insert with insert

Selection II

• E.coli with religated plasmid growth on medium containing antibiotics

• blue colonies due to active ßgalactosidase

• E.coli with plasmid and insert growth on medium containing antibiotics

• white colonies due to disrupted ßgalactosidase gene

• Mini (2 ml), Midi (50-200 ml), Maxi (>400 ml)

• harvesting of cells by centrifugation

• lyses of cells by basic pH (NaOH)

• neutralization by acetate

• on column washing

• elution of purified plasmid DNA with buffer or water

Preparation of plasmids

37°C

• cut with restriction enzyme

• agarose gel analysis:– size and conformation of nucleic acids in a sample

– approximate quantification of DNA

– separation and extraction of DNA fragments for recloning

• sequencing

Identification of plasmid DNA

advantages

• low costs

• time efficient

• high yield

• many systems with differentlyregulated promotors

disadvantages

• no posttranslationalmodification

• inclusion bodies

Bacterial expression systems

• “molecular farming”

• gene technology production of proteins from recombinant DNA

• expression vectors with poly linker site, marker and origin of replication

• initiation of transcription is regulated by distinct RNAP binding sites (promotor)

• translation dependent on binding sites for ribosomes (Shine-Dalgarno)

• E. coli regulation sequences need to be ahead of cloned gene

Expression of heterologous proteins in E. coli

• expression in inclusion bodies:

– higher amounts of protein– decreased proteolytic

degradation– easy to purify– refolding and purification steps time-consuming, loss of protein

• expression in membranes:

– lower expression level than in IB– “correct folding“ of the protein– lipid environment– challenge: protein purification

Expression of protein in E.coli

• in bacteria cytoplasmic aggregates of proteins

• usually sites of viral multiplication that consist of viral capsid proteins

• in human cells neuronal i.b. in disorders like Parkinson’s disease

• do not only contain misfolded protein

• results of expression of genes from an organism in another

• eucaryotic cDNA expressed as recombinant gene in procaryots risks the formation of inactive aggregates of proteins due to a foreign microenvironment (pH, osmolarity, folding mechanisms, processing systems)

inclusion bodies

different protein species

separation of cells fromsupernatant

diluted, low proteinyield

cell disruption

isolationrenaturation

renaturation problems, low ionic strength

cell disruption

lipids, proteases, high protein yield

separation of unsoluble material (cell membranes, cell walls,…)

chromatography

intracellular proteins inclusion bodies extracellular proteins

• cell suspensions are pressed through very narrow spaces

purification of proteins from inclusion bodies

cellbankampoulle

shaking flask100 – 500 ml

prefermenter50 l

production fermenter5,000 l

biomass harvesting

washing

cell breakage

harvest inclusion bodies

washing

purified inclusion bodies

Industrial isolation of inclusion bodies

cell disruption

• chemical (EDTA, lyzozyme, hypotonic solutions, Triton-X 100)

• freezing and thawing

• mortar and pestille

• vibration mills

• ultrasonics

• french press (small volume)

• Manton-Gaulin homogenator (V > 1 l)

• unphysiological environment inactivation, denaturation, proteolysis

• changes in pH buffer

• prevention of oxidation DTT, ß-mercaptoethanol (mM)

• complexation of metall ions EDTA

• low ionic strength NaCl, KCl (50 – 100 mM)

• aggregation of proteins Triton-X 100 (0.02%)

protection of proteins

• once from pancreas of cattle and pig– immunological reactions

– 50 pigs per diabetic person per year

• 1982 production in genetically engineered bacteria (NovoNordisk)

• today production in bacteria and yeast

Production of human insulin I

CUT

LIGATION

TRANSFORMATION

FERMENTATION

PURIFICATION

Production of human insulin II

• characteristics and „content“ of a plasmid

• transformation efficiency and antibiotic activity

• advantages and disadvantages of E.coli as an expression system

• purification of different protein species

Just to sum up…

E. coli is your friend