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ProteinCloning

Production

Purification Structural analysis:

• NMR

• X-ray

Protein/ligand interactions

Bioinformatics

RNA

Cell-free production

Alt.

Cpep10 course:Protein expression in E.coli

Problem 1: Structural integrity

Problem 3: Size

Problem 2: Space and time dependent interaction network

Problem 4: Unstructured pieces

Specials: Expression vectors, tags, cell-free systems

Problem 5: Codon usage

Change from native to host environment

The host

• Inexpensive• Easy to manipulate• Well characterized• Grows quickly• Recombinant protein up to 50% total protein• Easy and cheap labeling protocols for NMR and x-ray• Many expressionvectors and fusionproteins available• Simple mutagenesis and screening protocols• Use of cheap In-vitro transcription translation systems

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How complex it could be !!!Natural synthesis of actin

Polymerizes controlled

Binds number of proteinsnebulin/tropomyosintroponins/myosinthymosin/profilingelsolin/actinin ...

mRNA transport to specific location

Problem: folding pathway / controlled interactions

Specific eukaryotic chaperonepathway

Acetylated N-terminus

Where a lot of proteins interact !The muscle

• Strong interactions

• Strong forces

• Many interactions

• Highly regulated interactions

• Flexibility

Result of TAP-method analysis:Average number of interactionpartners of a given yeast proteinis 5 - 10

Problem: Interaction modi / multiple interactions

3 MegaDalton proteinstructureto solve: Titanic enterprise

Problem: size / unstructured pieces

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Typical medium sized protein

• Independent domains• Head to tail interaction• Posttranslational modification• Conformational change• Linear peptides• Protein/protein interaction• Protein/lipid interaction

insolubleinsoluble

Domain phasing

solublesoluble

insolubleinsoluble

N CDomainDomainboundariesboundariesare well are well defineddefined

Defining a domain withmultiple sequence alignment

Domain boundaries ofKH domain of FMRwas predicted.

Procaryotic membernusA stops her

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Expressing the KH domain defined bymultiple sequence alignment

After prediction ofdomain boundariesproduced proteinwas very unstableand lowexpressed.

More variants

Redefining a domain after expression

Domain boundaries ofKH domain afterexpression andsolving structure

Missing helix of KH domainPastore group, London

Domain phasing :Too much input

Multiple sequence alignment includes prokaryotic sequence with different topology .

Literature

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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.

Literature

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

insolubleinsoluble

Domain independent ?

solublesoluble

ADomain ADomain Aisisintegratedintegratedin structurein structure

ADomain ADomain Ais partiallyis partiallyindependentindependent

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Rule of thumb:Rule of thumb:

Good start : N-end ruleGood start : N-end rule55’’ codonscodons: AAA = K: AAA = K

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

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

???

• small scale production via autoinduction

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•• Creation of mutant libraryCreation of mutant library•• Random mutations / DNA shufflingRandom mutations / DNA shuffling•• Deletion seriesDeletion series

(like nuclease treated full length DNA)(like nuclease treated full length DNA)

•• Reporter proteinReporter protein•• Fusion to C-terminus of target proteinFusion to C-terminus of target protein•• If reporter folds it will give signalIf reporter folds it will give signal•• N- terminus should be foldedN- terminus should be folded

Is your protein folded ?

CAT (chloramphenicol res.)GFPComplementation methodsMarker genes / proteomics

Promoter PassengerCarrier

Vector backbone

origin resistance

The Vectors:Typical structure

Affinity_high stability_high production_cleavable

Expression story 1 :Extracellular Ig domain with disulfide bridge

Screening of Tags:

• GST• His• trx• dsbA

Screening of proteasecleavage sites :

• Thrombin• Tev protease

Result:dsbA Tev with low yieldbut folded

Leaderlessversion of dsbA

Strains withmutations in redoxsystem[Origami…]

unpublished

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2. Double Tag to get full length

Natural unstructured titin peptide [PEVK-element]

Labeit group, 1997Mannheim unpublished

Double Tag vector

PEVKPEVK

Publication on purification of arecombinant PEVK fragment

Why it’s important :

• Design might be wrong andpeptide is unfolded

• Design is OK and peptide isunstructured

Ligand interaction

3. Unstructured = Native ?3. Unstructured = Native ?

Kellenberger et al.,EMBL 2001

Why test multiple vectors :Yield

Carrierproteins + CH domains

Protease

Parallel preparation:

Uwe Sauer,Umea 1998-2006

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The VectorsThe Vectorsgene xgene x

gene xgene x

gene xgene x

gene xgene x

gene xgene x

Unpublished

Vector backgroundCloning advantage :

• Cut medium sized gene out

• Which produces colored protein

• False positives will be colored

Alternative if domain is well defined:MonoGFP fusion

Ni

Ni

Ni

Ni

Ni

Ni

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List of vectors

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pETM13N_HIS_NUSA_GSTEV_GFP

7559bp

Kan®

ori

T7

YFP

lacI

His6

Tev

nusA-Carrier

XhoI (158)

NotIBamHI

Acc65I (204)

NcoI (932)

XbaI

File with Map/Features/SequenceFeatures = Data file

• MW,pI• Gels • Purification data

Carrier His_GSTev/

N_His

His_PreScission/

N_His

His_Enterokinase/

N_His

His_Thrombin/

N_His

N_His

__ dir

C-His N-strep/

C-strep

C-fusion

Trx */* */* */ */ * *

GST */* */* */* */* * *

MBP */* */* * * * * - / *

DsbA */ */ * - / *

NusA */* */* * *

DsbC * * - / *

Ztag1

Ztag2 */* */* * * *

GB1 */* */* * * *

DsbAin * * *

DsbCin * * *

EFtag * * *

Mistic

ZZ tag * *

TolA

mGFP *

fGFP *

sMBP - / *

T4lyso *

Zr-pep

SF1pep

Webpages EMBL core facility,New KBC webpages Umeå

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

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Heterodimers of2 different complexes

One partner is tagged ,the other not.

+ + +

Max/Myc HNF1/DCoH

Coexpression : Results

Schallreuter et al,BBRC, 2003

Coexpression Coexpression

Two-plasmid

KanRori1

+AmpR

ori2

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]

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Old expression problem solved:Csk_src

Src tyrosine kinase insoluble

Src tyrosine kinase + Yersinia phosphatase 10% soluble and functional

Expression in Baculovirus infected insect cells and yeast

Levinson et al,

Where to go for coexpression ?• Lac repressor : pREP4

• T7 Lysozyme : pLysS

• rare tRNAs : CodonPlus strain

• Protein modifications :

• ASF/SF2 phosphorylated by SRPKinase 1

• Farnesyl group added by transferase (lipid group)

• Dephosphorylation of tyrosine kinases (abl, src etc) for better solubility

• Heterodimers to keep protein soluble: max/myc

• Chaperones groEL/ES to increase solubility

Message : Wrong partners - no folding - no expression system

Rare codon effectsTranscriptionfactor expressed in E.colicreates another subband

MW - + - + - + IPTG

ADD.PEPTIDE

FULL LENGTH

AGGAGG CGACGG ptRNA

Remenyi 2001,EMBL

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Rare codon effects:Misincorporation of Lysine

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

Pastore group, London

Codon Frequency in E. coli

Codon usage in E.coli :

Problems:

• Arg R• Ile I• Leu L

• Gly• Pro

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

Solutions:

• Codon plus / Rosetta strains• Patch rare codons: Partial gene synthesis• Scattered rare codons: Gene synthesis

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Cell-free expression

+ Nanodiscs and/or lipids Membrane protein

High copy number

Very pure

Kit

Apolipoprotein astool

Lessons from protein structures:Expression problems in E.coli

Multiple constructs/coexpression of modifiers

Cut in pieces and reassemble

Coexpression strategies or/and reassemble

Unstructured doesn’t mean unfolded or non native

Problem 1: Structural integrity

Problem 3: Size

Problem 2: Space and time dependent interaction network

Problem 4: Unstructured pieces

Parallel strategies in cloning, vector selection, strains, …..