Protein expression in E.coli: Lessons from structural biology Problem 1: Structural integrity Problem 3: Size Problem 2: Space and time dependent interaction network Problem 4: Unstructured pieces Specials: Expression vectors, NMR use, Tags Problem 5: Codon usage
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Protein expression in E.coli:Lessons from structural biology
Problem 1: Structural integrity
Problem 3: Size
Problem 2: Space and time dependent interaction network
Problem 4: Unstructured pieces
Specials: Expression vectors, NMR use, Tags
Problem 5: Codon usage
How complex it could be !!!Natural synthesis of actin
Polymerizes controlled
Binds number of proteinsnebulin/tropomyosintroponins/myosinthymosin/profilingelsolin/actinin ...
Multiple sequence alignment includes prokaryotic sequence with different topology .
Domain phasing :KH domain +
Qua2 region :Additional 10 aa secondary structure element is important for specific binding to ss branchpoint RNA .
Domain phasing :The missing domain
Complex domaininterface
No independence
Domain interphasesvary and change inother structuralcontext
Attempts to express the PH domain (443-551) in E. coli were notsuccessful, and the protein product is insoluble.An extension to the N terminus with a small part of DbH domain(422-551) is soluble.
Unique fold has not too much meaning for expressionresult !
protein function is unique
protein context of isolated domain is unique
PH domains with unique foldPH domains with unique fold
SOS ph domain BTK ph domain
Get rid of flexible bits
FERM structure:N and C-termini come together
• Flexible central bit removed• N- and C- terminal pieces independent
expressed• Reassembled complex
X-raysamples
N-WASP EVH1 Domain Sequence
• Domain construct insoluble• Constructs of domain fused to a minimal binding peptide
via a (Gly-Ser-Gly-Ser-Gly) linkers• One version yielded highly soluble protein
Physical fusion of a Physical fusion of a ligandligand
start and endstart and endhydrophilichydrophilicsecondary structuresecondary structurenext neighbour domainnext neighbour domain
full length limited proteolysisfull length limited proteolysis
Are there simple rules ?
A
Message : Wrong borders - no folding - no expression system
Tag or no tag ?Tag or no tag ?Problems of dimeric Tag(GST)
N
N
domain or C-terminal piecesmissing due todegradation,translational stops etc.
Domain phasing :Multiple constructs
solublesoluble
N C
Staggered oligosThe winningteam
Typical weekTypical week
X-talsX-tals
•• multiple multiple PCRs PCRs and vectorsand vectors •• scale up scale up
• N15 probe
•• solubility screen solubility screen
???
339-416 345-404345-416 339-404
NMR screening of domain boundariesNMR screening of domain boundaries
N and C-termini are now in good shape
SMN tudor domain
X-ray construct
•• Creation of mutant libraryCreation of mutant libraryrandom mutations / DNA shufflingrandom mutations / DNA shufflingdeletion seriesdeletion series
like nuclease treated full length DNAlike nuclease treated full length DNA
•• Reporter proteinReporter proteinfusion to C-terminus of target proteinfusion to C-terminus of target proteiniF iF reporter folds it will give signalreporter folds it will give signalN- terminus should be foldedN- terminus should be folded
Is your protein folded ?
CATGFPComplementationMarker genes / proteomics
Combinatorial libraries together with reporter
PCR via multiplephased primers
Enzymatic orphysical breaks
Nucleasetruncation
Error prone PCR
Mutate andDNA shuffling
Reporter fusions
Coexpression : Inclusion bodies
Myc doesn’t form homodimers likemax and expresses in inclusionbodies in E.coli.
Very hydrophobicinterface
Coexpression : No inclusion bodies
Myc forms stable heterodimer withmax and expresses soluble in thecomplex.
Very hydrophobicinterface
Transcriptional Transcriptional coactivatorcoactivatorfails to interact withfails to interact withtranscription factor in tubetranscription factor in tube
Coexpression : Protein association only in vivo
E.colior cotranslation
Dcoh
+
HNF1 Complex
Heterodimers of2 different complexes
One partner is tagged ,the other not.
+ + +
Max/Myc HNF1/DCoH
Coexpression : Results
Coexpression Coexpression
Two-plasmid
KanRori1+
AmpRori2
gene I gene II
Coexpression Coexpression Dicistronic
XbaI SpeISD gene I
SD gene IIXbaI
T7
gene I gene II
+ Rnase deficient strainBL21Star
Advantage in cotranslational folding:Assembly of 7 nucleoporins into 0.5 MDComplex [Lutzmann et al]
Coexpression varies
Dicistronic variations
Staggered distances of 2nd translation initiation site
+/ - + + +
Where to go for coexpression ?• Lac repressor : pREP4
• T7 Lysozyme : pLysS
• rare tRNAs : CodonPlus strain
• Protein modification :
• ASF/SF2 phosphorylation by SRPK1
• Farnesyl group by transferase
• Heterodimers max/myc HNF1/DCoH
• Chaperones groEL/ES increases solubility of csk
• TEV protease in MBP Tev fusions increases solubility ofpassenger protein Message : Wrong partners - no folding - no expression system
FMR KH domainshows strange 28 Ddifferent species in mass
ADD.PEak
FULL LENGTH
Reason:Rare arginine codonsAGAs or AGGs areloaded with lysine tRNAs;MW difference of 28 D
Rare codon effects:Proteolysis
• 2 central consecutive rare codons cause very low expression levelof Tev protease
• Causes processive degradation of nascent polypeptide at slowed translation point
……AGGAGG……. 49 50
……AGGAGG
Ribosome falls off
Proteolysis coupled to translational pausing
Codon usage problems
Signs:
• Mass difference AGA loads AAA [K] / CGG loads CAG [Q]• Consecutive rare codon spots• Protein ladder after purification with N-tag or• No expression• Signs of toxicity
• Library of FNIII domain scaffold on surface loop• Phage display or yeast two hybrid with target• Matrix with monobody
• Endoribonuclease ACA specific• Change gene to non ACA codon usage by gene synthesis• Induce nuclease in production phase• No production of E.coli proteins
Single protein production systems
Future of E.coli :
Labeling techniques• Specific and unnatural aminoacids• NMR• D2O
Posttranslational modificationsIntein ligationLibrary and screen methodsIncell NMRSPP : Single protein production systemsMonobodies for target specific affinity columns
Physical fusion of a modified Physical fusion of a modified ligandligand
Domain or protein interaction but affinity too low;useful with modified peptides or regulatorypeptides