Novel Technologies for Recombinant Protein Overexpression in Escherichia coli

Tobias Cornvik

Department of Biochemistry and Biophysics

Stockholm University

2006=

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Abstract qÜÉ=ìëÉ=çÑ=êÉÅçãÄáå~åí=éêçíÉáå=áë=~=ÅçêåÉêëíçåÉ=áå=ã~åó=ëíêìÅíìê~ä=

~åÇ= ÑìåÅíáçå~ä= ëíìÇáÉëK=qÜÉ=ÉåíÉêáÅ=Ä~ÅíÉêáìã=bëÅÜÉêáÅÜá~=`çäá= áë= íÜÉ=

ãçëí= Åçããçåäó= ìëÉÇ= çêÖ~åáëã= Ñçê= éêçÇìÅáåÖ= êÉÅçãÄáå~åí= éêçíÉáåëK=bK= Åçäá= Ü~ë= ëÉîÉê~ä= ~Çî~åí~ÖÉë= çîÉê= çíÜÉê= ÉñéêÉëëáçå= ÜçëíëI= Äìí= ~äëç=

çåÉ=ã~àçê=Çáë~Çî~åí~ÖÉ=J=íÜÉ=éêçíÉáå=çÑ=áåíÉêÉëí=ÇçÉë=åçí=~äï~óë=~Ççéí=áíë=å~íáîÉ=ÅçåÑçêã~íáçåK=fåëíÉ~Ç=íÜÉ=éêçíÉáå=ãáÖÜí=Ñçêã=ä~êÖÉ=áåëçäìÄäÉ=

~ÖÖêÉÖ~íÉëI= áåÅäìëáçå= ÄçÇáÉëI= ïáíÜáå= íÜÉ= ÅÉääK= få= é~êíáÅìä~êI= íÜÉ=ÜÉíÉêçäçÖçìë=çîÉêÉñéêÉëëáçå=çÑ=Éìâ~êóçíáÅ=~åÇ=ãÉãÄê~åÉ=éêçíÉáåë=~êÉ=

íêçìÄäÉëçãÉK=få=íÜáë=íÜÉëáëI=ãÉíÜçÇë=~êÉ=ÇÉëÅêáÄÉÇ=íÜ~í=Å~å=ÄÉ=ìëÉÇ=íç=áåÅêÉ~ëÉ=íÜÉ=

äáâÉäáÜççÇ=çÑ=çîÉêÉñéêÉëëáåÖ=Éìâ~êóçíáÅ=éêçíÉáåë=~ë=ïÉää=~ë=ãÉãÄê~åÉ=éêçíÉáåëK= få= é~êíáÅìä~êI= ~= åçîÉä= ãÉíÜçÇ= áë= ÇÉëÅêáÄÉÇ= íÜ~í= Å~å=ÇáëíáåÖìáëÜ= ÄÉíïÉÉå= Ä~ÅíÉêá~ä= ÅçäçåáÉë= ÉñéêÉëëáåÖ= ëçäìÄäÉ= éêçíÉáåë=

Ñêçã= íÜçëÉ= ÉñéêÉëëáåÖ= áåÅäìëáçå=ÄçÇáÉëK=qÜÉ=ãÉíÜçÇ=ìíáäáòÉë= íÜÉ= Ñ~Åí=íÜ~í=áåÅäìëáçå=ÄçÇáÉë=~êÉ=çÑ=~=ÅçåëáÇÉê~ÄäÉ=ëáòÉ=~åÇ=Å~å=ÄÉ=êÉãçîÉÇ=Äó=

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List of papers qÜáë=íÜÉëáë=áë=Ä~ëÉÇ=çå=íÜÉ=ÑçääçïáåÖ=é~éÉêëI=êÉÑÉêêÉÇ=íç=áå=íÜÉ=íÉñí=Äó=íÜÉáê=oçã~å=åìãÉê~äëK=

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Abbreviations Åak^= = `çãéäÉãÉåí~êó=ak^=`çcá=Ääçí= = `çäçåó=cáäíê~íáçå=_äçí=

`JíÉêãáåìë= = `~êÄçñóJqÉêãáåìë=aaj== = åJaçÇÉÅóä=≈JaJã~äíçëáÇÉ=

bj= = = bäÉÅíêçå=jáÅêçëÅçéó=cáaç= = = cáäíê~íáçå=açí=_äçí=

dcm= = = dêÉÉå=cäìçêÉëÅÉåí=mêçíÉáå=dpq= = = däìí~íÜáçåÉ=ëJqê~åëÑÉê~ëÉ=

fj^`= = fããçÄáäáòÉÇ=jÉí~ä=^ÑÑáåáíó=`Üêçã~íçÖê~éÜó=j_m= = = j~äíçëÉ=_áåÇáåÖ=mêçíÉáå=

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m`o= = = mçäóãÉê~ëÉ=`Ü~áå=oÉ~Åíáçå=pd= = = píêìÅíìê~ä=dÉåçãáÅë=

qou= = = qÜáçêÉÇçñáå=tq= = = táäÇ=qóéÉ=

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Introduction qÜÉ=Üìã~å=ÄçÇó=Åçåëáëíë=çÑ=~ééêçñáã~íÉäó=NMM=íêáääáçå=ENMNQF=ÅÉääë=~åÇ=~äëç= Üçëíë= ~åçíÜÉê= èì~Çêáääáçå= ENMNRF= ÅÉääë= áå= íÜÉ= Ñçêã= çÑ= ãáÅêçJ

çêÖ~åáëãë= íÜ~í= äáîÉ= áå=çìê=Öìí= xNzK=b~ÅÜ=ÅÉää= áë= ëìêêçìåÇÉÇ=Äó=~=îÉêó=íÜáå= Ñáäã= çÑ= äáéáÇë= EÑ~íëF= ëÜáÉäÇáåÖ= íÜÉ= áåíÉêáçê= EÅóíçéä~ëãF= Ñêçã= íÜÉ=

ëìêêçìåÇáåÖ=ãÉÇá~K=fåëáÇÉ=íÜÉ=ÅÉää=~êÉ=~=ãóêá~Ç=çÑ=ÇáÑÑÉêÉåí=ãçäÉÅìäÉëX=ëìÖ~êëI= äáéáÇëI= åìÅäÉçíáÇÉë= EíÜÉ= ÄìáäÇáåÖ= ÄäçÅâë= çÑ= ak^= ~åÇ= ÉåÉêÖó=

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=Table 1 The genetic code. Nucleotides are shown as their single letter code and the amino acid each triplet codes for is shown to the right. The genetic code is conserved

between different species. A gene coding for a human protein can therefore be transferred to a bacterium and still be correctly translated.

=

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= NP

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=

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Deposited structures for the yearTotal available structures

=Figure 1 Number of structures deposited in the Protein Data Bank. Data obtained from the PDB website.

=

Membrane Proteins ^ää=äáîáåÖ=ÅÉääë=~êÉ=ëìêêçìåÇÉÇ=Äó=~=íÜáå=ãÉãÄê~åÉ=ÅçãéçëÉÇ=çÑ=äáéáÇëK=qÜÉêÉ= Ü~îÉ= ÄÉÉå= ëÉîÉê~ä= ÇáÑÑÉêÉåí= ãçÇÉäë= çÑ= Üçï= íÜáë= ãÉãÄê~åÉ= áë=

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=========================================O=ÜííéWLLïïïKéÇÄKçêÖ=

= NQ

EcáÖìêÉ=OFK= få=NVTO=páåÖÉê= ~åÇ=káÅÜçäëçå=éêçéçëÉÇ= íÜÉ= ÑäìáÇ=ãçë~áÅ=ãçÇÉäI=ïÜáÅÜ=ïáíÜ=ëçãÉ=ãçÇáÑáÅ~íáçåëI=áë=ëíáää=íçÇ~ó=íÜÉ=ãçÇÉä=ìëÉÇ=íç=ÇÉëÅêáÄÉ= ÄáçäçÖáÅ~ä= ãÉãÄê~åÉë= xOSzK= qÜáë= ãçÇÉä= êÉí~áåë= íÜÉ= äáéáÇ=

Äáä~óÉêI= Äìí= íÜÉ= éêçíÉáåJäáéáÇJéêçíÉáå= ë~åÇïáÅÜ= ï~ë= êÉéä~ÅÉÇ= Äó= ~=ãçÇÉä=ïÜÉêÉ=íÜÉ=éêçíÉáåë=“Ñäç~íÒ=áå=íÜÉ=ãÉãÄê~åÉ=EcáÖìêÉ=OFK===

Danielli and Davson 1935 Singer and Nicolson 1972

Protein Integral protein

Peripheral proteina b

= Figure 2 Schematic outline of the different lipid bilayer models. (A) The model

suggested by Danelli and Davson. In this model the lipid is organised in a bilayer surrounded by a protein layer. (B) The fluid mosaic model suggested by Singa and

Nicolson. The lipids are arranged in bilayer but the proteins can span the bilayer.

=

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qÜÉ=ÄáåÇáåÖ=çÑ=aå~h= íç= íÜÉ=éçäóéÉéíáÇÉ= ÅÜ~áå= áë= ÅçìéäÉÇ=ïáíÜ=^qm=ÜóÇêçäóëáë= Äó= aå~hK= qÜÉ= ëìÄëíê~íÉ= áë= êÉäÉ~ëÉÇ= Ñêçã= aå~h= Äó= íÜÉ=

ÉñÅÜ~åÖÉ=çÑ=^am=íç=^qmI=ÅçåíêçääÉÇ=Äó=íÜÉ=dêéb=éêçíÉáåK=tÜÉå=íÜÉ=éêçíÉáå=áë=êÉäÉ~ëÉÇI=áí=ãáÖÜí=êÉ~ÅÜ=áíë=å~íáîÉ=ÅçåÑçêã~íáçå=çê=ìåÇÉêÖç=~=ëÉêáÉë=çÑ=aå~h=ÅóÅäÉëK=qÜÉ=éçäóéÉéíáÇÉ=ÅÜ~áå=ãáÖÜí=~äëç=ÄÉ=í~êÖÉíÉÇ=íç=

íÜÉ= dêçbiJdêçbp= ÅÜ~éÉêçåáå= ëóëíÉãK= dêçbi= áë= ~= îÉêó= ä~êÖÉ= EúUMM=âa~F=éêçíÉáå=ÅçãéäÉñ=ÅçåëáëíáåÖ=çÑ= íïç=ëí~ÅâÉÇ=ÜÉéí~ãÉêáÅ=êáåÖëK= få=áíë= çéÉå= ÑçêãI= íÜÉ= ÅÉåíê~ä= Å~îáíó= çÑ= dêçbi= áë= äáåÉÇ= ïáíÜ= ÉñéçëÉÇ=

ÜóÇêçéÜçÄáÅ= ~ãáåç= ~ÅáÇëI= Ñ~Åáäáí~íáåÖ= íÜÉ= ÄáåÇáåÖ= íç= é~êíäó= ÑçäÇÉÇ=éçäóéÉéíáÇÉëK= réçå= ÄáåÇáåÖ= çÑ= dêçbp= íç= dêçbiI= ^qm= ÜóÇêçäóëáë=

çÅÅìêë= ~åÇ= íÜÉ= ÅçãéäÉñ= ìåÇÉêÖçÉë= ~= ä~êÖÉ= ÅçåÑçêã~íáçå~ä= ÅÜ~åÖÉ=ïÜáÅÜ= äÉ~Çë=íç=íÜÉ=“êÉãçî~äÒ=çÑ= íÜÉ=ÜóÇêçéÜçÄáÅ=~ãáåç=~ÅáÇë= äáåáåÖ=íÜÉ=Å~îáíó=ï~ääK=fí=áë=áå=íÜáë=éêçÅÉëë=íÜ~í=íÜÉ=éêçíÉáå=áåëáÇÉ=íÜÉ=dêçbiJ

dêçbp= ÅçãéäÉñ= ÑçäÇë= xRMzK= eçïÉîÉêI= ãçëí= çÑ= íÜÉ= éêçíÉáåë= êÉäÉ~ëÉÇ=Ñêçã= dêçbiJdêçbp= ~êÉ= åçí= áå= íÜÉ= å~íáîÉ= ëí~íÉ= ~åÇ= ~êÉ= íÜÉêÉÑçêÉ=

ëìÄàÉÅíÉÇ=íç=~åçíÜÉê=êçìåÇ=çÑ=ÑçäÇáåÖ=áå=íÜÉ=ÅÜ~éÉêçåáåK===

Holding Chaperones réçå= ÉåîáêçåãÉåí~ä= ëíêÉëëI= ëìÅÜ= ~ë= íÉãéÉê~íìêÉ= çê= çñáÇ~íáîÉ= ëíêÉëëI=

ÜçäÇáåÖ=ÅÜ~éÉêçåÉë=ÄáåÇ= íç=é~êíäó=ìåÑçäÇÉÇ=éêçíÉáåëI=ïÜáÅÜ=éêÉîÉåíë=íÜÉ= ìåÑçäÇÉÇ= éêçíÉáåë= Ñêçã=~ÖÖêÉÖ~íáåÖ= xQUz= EcáÖìêÉ= PFK=qÜÉ= ÜçäÇáåÖ=

ÅÜ~éÉêçåÉë= Çç= åçí= ~ÅíáîÉäó= ~ëëáëí= áå= íÜÉ= ÑçäÇáåÖ= çÑ= íÜÉ= éêçíÉáåI= Äìí=áåëíÉ~Ç=é~ëë= íÜÉ=éêçíÉáå= íç= íÜÉ=aå~hLaå~g=çê=dêçbiJdêçbp=ëóëíÉãë=ïÜÉå=íÜÉ=ëíêÉëë=Ü~ë=ÄÉÉå=êÉäáÉîÉÇK=eçïÉîÉêI= áÑ= íÜÉ=ëíêÉëë=ÅçåÇáíáçå= áë=

éêçäçåÖÉÇI=íÜÉ=ÅÜ~éÉêçåÉ=ëóëíÉãë=~êÉ=ë~íìê~íÉÇ=~åÇ=íÜÉ=Ñçêã~íáçå=çÑ=áåÅäìëáçå=ÄçÇáÉë=áë=áåÉîáí~ÄäÉK=

=

= ON

Disaggregating Chaperones bîÉå= áÑ= áåÅäìëáçå= ÄçÇáÉë= ~êÉ= ÑçêãÉÇI= íÜÉêÉ= ~êÉ= ÅÜ~éÉêçåÉë= íÜ~í= Å~å=Çáë~ÖÖêÉÖ~íÉ= éêçíÉáåë= Ñêçã= íÜÉ= áåÅäìëáçå= ÄçÇáÉë= EcáÖìêÉ= PFK= qÜáë=éêçÅÉëë=áë=ã~áåäó=éÉêÑçêãÉÇ=Äó=íÜÉ=`äé_=ÅÜ~éÉêçåÉ=xQUzK=få=~å=^qmJ

ÇÉéÉåÇÉåí= éêçÅÉëëI= `äé_= ëçäìÄáäáëÉë= éçäóéÉéíáÇÉë= Ñêçã= íÜÉ= áåÅäìëáçå=ÄçÇáÉëI=ÜçïÉîÉê=íÜÉ=êÉÑçäÇáåÖ=çÑ=íÜÉ=éêçíÉáåë=êÉèìáêÉë=íÜÉ=aå~hLaå~g=

ëóëíÉãK= fí= Ü~ë= ~äëç=ÄÉÉå= ëÜçïå= íÜ~í=aå~hLaå~g= Å~å= ëçäìÄáäáëÉ= ëã~ää=~ÖÖêÉÖ~íÉë=xRNzK==

=

GroEL

GroES

ClpE

DnaK

DnaJ

TF

DnaK

DnaJ

Holding Chaperones

Correctly folded protein

Aggregated Protein / Inclusion bodies

==Figure 3 Schematic outline of the folding process in E. coli and the chaperones involved. When a nascent chain exits the ribosome it can either fold spontaneously

into its correct conformation or be a target for chaperones. The polypeptide might also aggregate into inclusion bodies. Arrows indicate the possible pathways and

chaperones the polypeptide may encounter.

=

=

= OO

Membrane Proteins jÉãÄê~åÉ=éêçíÉáåë=Ü~îÉ= íç= í~ÅâäÉ=~=ÇáÑÑÉêÉåí=âáåÇ=çÑ=éêçÄäÉã=ïÜÉå=

íÜÉó=ÑçäÇX=íÜÉó=ãìëí=áåëÉêí=áåíç=íÜÉ=äáéáÇ=Äáä~óÉêK=qÜÉ=ÑçäÇáåÖ=éêçÅÉëë=çÑ=ãÉãÄê~åÉ=éêçíÉáåë= Å~å=ÄÉ= ÇáîáÇÉÇ= áåíç= íïç= ëíÉéëI= íÜÉ= áåëÉêíáçå= áåíç=íÜÉ= ãÉãÄê~åÉ= ~åÇ= íÜÉ= é~ÅâáåÖ= çÑ= íÜÉ= íê~åëãÉãÄê~åÉ= ÜÉäáÅ~ä=

ëÉÖãÉåíëK==

Insertion into the Membrane cçê=ãçëíI=áÑ=åçí=~ää=ãÉãÄê~åÉ=éêçíÉáåëI=íÜÉ=íê~åëãÉãÄê~åÉ=ëÉÖãÉåíë=~êÉ= ~ÅíáîÉäó= áåëÉêíÉÇ= áåíç= íÜÉ= äáéáÇ= Äáä~óÉê= ïáíÜ= íÜÉ= ÜÉäé= çÑ= éêçíÉáå=ã~ÅÜáåÉêóK= cçê= ~= ä~êÖÉ= ã~àçêáíó= çÑ= íÜÉ= bK= Åçäá= áåíÉÖê~ä= ãÉãÄê~åÉ=

éêçíÉáåë=EfjmF=íÜáë=éêçíÉáå=ã~ÅÜáåÉêó=áë=íÜÉ=pÉÅJíê~åëäçÅçåK===qÜÉ= áåëÉêíáçå= éêçÅÉëë= íóéáÅ~ääó= ëí~êíë= ïáíÜ= ÄáåÇáåÖ= çÑ= íÜÉ= ëáÖå~ä=

êÉÅçÖåáíáçå= é~êíáÅäÉ= EpomF= íç= ~äéÜ~= ÜÉäáÅ~ä= ÜóÇêçéÜçÄáÅ= ëíêÉíÅÜÉë= çÑ=~ãáåç=~ÅáÇë=~ë=íÜÉó=Éñáí=íÜÉ=êáÄçëçãÉ=xROzK=qÜÉ=ÄáåÇáåÖ=çÑ=íÜÉ=pom=íç=íÜÉ= å~ëÅÉåí= ÅÜ~áåë= äÉ~Çë= íç= ~= íê~åëäçÅ~íáçå= çÑ= íÜÉ= êáÄçëçãÉ= íç= íÜÉ=

ãÉãÄê~åÉ=ïÜÉêÉ=pom=ÄáåÇë=íç=íÜÉ=pom=êÉÅÉéíçêK===

SRP

RibosomeRibosome

Ribosome

SRP

SRP

==Figure 4 A schematic outline of the membrane protein biosynthesis. As the nascent

chain of membrane protein exits the ribosome, the signal recognition particle (SRP)

binds to alpha helical hydrophobic segments. This leads to a translocation of the ribosome to the translocon where the transmembrane segments are transferred

laterally into the lipid bilayer.

= OP

qÜÉ=pom=êÉÅÉéíçê=áë=~ëëçÅá~íÉÇ=íç=íÜÉ=pÉÅ=íê~åëäçÅçåI=ïÜáÅÜ=êÉëáÇÉë=áå=íÜÉ=áååÉê=ãÉãÄê~åÉ=çÑ=bK=ÅçäáK=fÑ=äçåÖ=ÉåçìÖÜ=ÜóÇêçéÜçÄáÅ=ëÉÖãÉåíë=~êÉ= ÉåÅçìåíÉêÉÇ= áå= íÜÉ= pÉÅ= íê~åëäçÅçåI= íÜÉëÉ= ~êÉ= íê~åëÑÉêêÉÇ= ä~íÉê~ääó=

áåíç= íÜÉ=ãÉãÄê~åÉK=^= ëÅÜÉã~íáÅ= çìíäáåÉ= çÑ= íÜáë= éêçÅÉëë= áë= ëÜçïå= áå=cáÖìêÉ=QK====

Packing of Transmembrane Helices qÜÉ=ÑçäÇáåÖ=çÑ=íÜÉ=íê~åëãÉãÄê~åÉ=éêçíÉáåë=ÇáÑÑÉêë=Ñêçã=íÜ~í=çÑ=ëçäìÄäÉ=éêçíÉáåë=åçí=çåäó=áå=íÜ~í=íÜÉó=Ü~îÉ=íç=ÄÉ=áåëÉêíÉÇ=áåíç=íÜÉ=ãÉãÄê~åÉI=

Äìí= ~äëç= ÄÉÅ~ìëÉ= íÜÉ= ëÉÅçåÇ~êó= ëíêìÅíìê~ä= ÉäÉãÉåíë= çÑ= íÜÉ=íê~åëãÉãÄê~åÉ= éêçíÉáåë= ~êÉ= ÑçêãÉÇ= ÇìêáåÖ= íÜÉ= áåëÉêíáçå= áåíç= íÜÉ=

ãÉãÄê~åÉK= qÜÉ= é~êíäó= ìåÑçäÇÉÇ= ëí~íÉ= çÑ= ~= íê~åëãÉãÄê~åÉ= éêçíÉáå=Åçåí~áåë= ~= ÜáÖÜ= ÇÉÖêÉÉ= çÑ= ëÉÅçåÇ~êó= ëíêìÅíìêÉ= ëáåÅÉ= íê~åëãÉãÄê~åÉ=

ÜÉäáÅÉë=Ü~îÉ=ÄÉÉå= ÑçêãÉÇ= áå= íÜÉ= áåëÉêíáçå=éêçÅÉëë= xRPzK=`çãé~êÉÇ= íç=íÜÉ= ÑçäÇáåÖ= çÑ= ëçäìÄäÉ= éêçíÉáåëI= íÜÉ= ÑçäÇáåÖ= çÑ=ãÉãÄê~åÉ= éêçíÉáåë= áë=

äÉëë= ïÉää= ìåÇÉêëíççÇK= ^ë= ÇáëÅìëëÉÇ= ~ÄçîÉI= íÜÉ= ÜóÇêçéÜçÄáÅ= ÉÑÑÉÅí= áë=çåÉ=çÑ=íÜÉ=ÇêáîáåÖ=ÑçêÅÉë=áå=íÜÉ=ÑçäÇáåÖ=çÑ=ëçäìÄäÉ=éêçíÉáåëK=qÜáë=ÑçêÅÉ=

áë= ãçêÉ= çê= äÉëë= åçåJÉñáëíÉåí= ïáíÜáå= íÜÉ= ~ÅóäJÅÜ~áå= êÉÖáçå= çÑ= íÜÉ=ãÉãÄê~åÉ= xRPzI= áåëíÉ~Ç= çíÜÉê= ÑçêÅÉë= ÅçãÉ= áåíç= éä~óK= fí= áë= ÖÉåÉê~ääó=

ÅçåëáÇÉêÉÇ=íÜ~í=çåÉ=çÑ=íÜÉ=ã~áå=ÇêáîáåÖ=ÑçêÅÉë=ÄÉÜáåÇ=íÜÉ=é~ÅâáåÖ=çÑ=íÜÉ=íê~åëãÉãÄê~åÉ=ÜÉäáÅÉë= áë= íÜÉ= Ñ~îçê~ÄäÉ= áåíÉê~Åíáçå=ÄÉíïÉÉå=éçä~ê=êÉëáÇìÉë=xRQzK=qÜáë=áë=ëÜçïå=Äó=íÜÉ=Ñ~Åí=íÜ~í=íÜÉ=áåíÉêáçêë=çÑ=ãÉãÄê~åÉ=

éêçíÉáåë=~êÉ=ãçêÉ=éçä~ê=íÜ~å=íÜÉ=ÉñíÉêáçêë=xRRzK=eóÇêçÖÉå=ÄçåÇë=Ü~îÉ=íÜÉ=éçíÉåíá~ä=íç=ÄÉ=îÉêó=ëíêçåÖ=áå=íÜÉ=~éçä~ê=êÉÖáçå=çÑ=íÜÉ=äáéáÇ=Äáä~óÉê=xRSzK= péÉÅáÑáÅ= î~å= ÇÉê=t~~äë= áåíÉê~Åíáçåë= ~í= ÅäçëÉ= Çáëí~åÅÉë= éä~ó= ~å=

ÉëëÉåíá~ä= êçäÉ= ïÜÉå= é~ÅâáåÖ= íÜÉ= íê~åëãÉãÄê~åÉ= ÜÉäáÅÉëK= qÜÉ= ~ãáåç=~ÅáÇ= êÉëáÇìÉë= ~êÉ= åçí= íÜÉ= çåäó= Ñ~Åíçêë= íÜ~í= ~êÉ= áãéçêí~åí= Ñçê= íÜÉ=

ÑçäÇáåÖ= ~åÇ= áåëÉêíáçå= áåíç= íÜÉ= ãÉãÄê~åÉK= qÜÉ= äáéáÇë= ~åÇ= íÜÉ= äáéáÇ=Åçãéçëáíáçå=áå=íÜÉ=Äáä~óÉê=Ü~îÉ=ÄÉÉå=ëÜçïå=íç=áåÑäìÉåÅÉ=íÜÉ=áåëÉêíáçå=xRTI=RUz=~åÇ=Å~å=~äíÉê=íÜÉ=ÑçäÇáåÖ=é~íÜï~ó=xRVzK==

=

= OQ

Inclusion bodies ^ë= ÇáëÅìëëÉÇ= ~ÄçîÉI= íÜÉ= ÑçäÇáåÖ= çÑ= ~= éçäóéÉéíáÇÉ= áåíç= áíë= å~íáîÉ=

ÅçåÑçêã~íáçå= áë=åçí=~=íêáîá~ä= í~ëâ=áå=íÜÉ=ÅêçïÇÉÇ=bK=Åçäá=ÅÉää=~åÇ=íÜÉ=Ñçêã~íáçå=çÑ=áåÅäìëáçå=ÄçÇáÉë=áë=åçí=ìåÅçããçå=ïÜáäÉ=çîÉêÉñéêÉëëáåÖ=

~=éêçíÉáå=áå=bK=ÅçäáK=fåÅäìëáçå=ÄçÇáÉë=Å~å=î~êó=áå=ëáòÉ=~åÇ=ëÜ~éÉ=Äìí=íÜÉ=~ééêçñáã~íÉ=ã~ñáãìã= äÉåÖíÜ=çÑ= ~å= áåÅäìëáçå=ÄçÇó= áë=N=”ã=~åÇ= íÜÉ=ã~ñáãìã= îçäìãÉ= áë= êçìÖÜäó= MKS= ”ãP= xSMJSOzK= få= ÖÉåÉê~äI= íÜÉ= í~êÖÉí=

éêçíÉáå=ÅçåëíáíìíÉë=UMJVRB=çÑ=íÜÉ=ã~íÉêá~ä=áå=íÜÉ=áåÅäìëáçå=ÄçÇáÉë=xQUz=~åÇ= áë= ÅçãéäÉíÉäó= çê= é~êíäó= ìåÑçäÇÉÇK= eçïÉîÉêI= íÜÉêÉ= ~êÉ= Éñ~ãéäÉë=

ïÜÉêÉ= ÅçêêÉÅíäó= ÑçäÇÉÇ= ~åÇ= ~ÅíáîÉ= éêçíÉáåë= Ü~îÉ= ÄÉÉå= ÑçìåÇ= áå=áåÅäìëáçå=ÄçÇáÉë= xSPzK= píêìÅíìê~ä= ÖÉåçãáÅë=éêçÖê~ãë=Ü~îÉ= ëÅêÉÉåÉÇ=~=îÉêó= ä~êÖÉ= åìãÄÉê= çÑ= éêçíÉáåë= Ñçê= ëçäìÄäÉ= ÉñéêÉëëáçåK= fí= áë= íÜÉêÉÑçêÉ=

éçëëáÄäÉ= íç= çÄí~áå= ÖççÇ= ëí~íáëíáÅë= çå= íÜÉ= äáâÉäáÜççÇ= çÑ= éêçÇìÅáåÖ=ëçäìÄäÉ=éêçíÉáå=áå=bK=ÅçäáK=cçê=éêçâ~êóçíáÅ=çêÖ~åáëãë=íÜÉ=äáâÉäáÜççÇ=áë=~ééêçñáã~íÉäó= RMBS= ïÜáäÉ= Ñçê= Éìâ~êóçíáÅ= éêçíÉáåë= íÜÉ= ëìÅÅÉëë= ê~íÉë=

~êÉ= çå= íÜÉ= çêÇÉê= çÑ= OMJPMBSK= eçïÉîÉêI= íÜÉ= ëí~íáëíáÅë= Ñçê= íÜÉ=Éìâ~êóçíáÅ= éêçíÉáåë= áå= íÜáë= ëÉí= ãáÖÜí= ÄÉ= ëäáÖÜíäó= ëâÉïÉÇ= íçï~êÇë=

ëã~ääÉê=éêçíÉáåë=ëáåÅÉ=kjo=Ü~ë=ÄÉÉå=ìëÉÇ=áå=ãçëí=Å~ëÉëK=få=~=ëíìÇó=Äó=iì~å= Éí= ~ä= ~= îÉêó= ä~êÖÉ= Ñê~Åíáçå= çÑ= ~ää= locë= Ñêçã `~ÉåçêÜ~ÄÇáíáë=ÉäÉÖ~åëI= ~= ëã~ää= ïçêãI= ï~ë= ëÅêÉÉåÉÇ= Ñçê= ÉñéêÉëëáçå= áå=bK= Åçäá= xSQzK=

lîÉê~ää=NIRPS=çìí=çÑ=NMINST=ENRBF=ÅçìäÇ=ÄÉ=ÉñéêÉëëÉÇ=ëçäìÄäóK===

Degradation ^åçíÜÉê= ëíêÉëë= êÉëéçåëÉ= áå= bK= ÅçäáI= ÄÉëáÇÉë= áåÅäìëáçå= ÄçÇáÉëI= áë=áåÅêÉ~ëÉÇ=áå=îáîç=éêçíÉçäóëáë=xSRzK=mêçíÉçäóëáë=~ääçïë=íÜÉ=ÅÉää=íç=êÉÅóÅäÉ=

~ãáåç=~ÅáÇë=~åÇ=~îçáÇ=~ÅÅìãìä~íáçå=çÑ=~Äåçêã~ä=éêçíÉáåë=ïáíÜáå=íÜÉ=ÅÉää=xQUzK=bìâ~êóçíáÅ=ãìäíáJÇçã~áå=éêçíÉáåë=~êÉ=ÉëéÉÅá~ääó=ëÉåëáíáîÉ=íç=

éêçíÉ~ëÉë=ëáåÅÉ=íÜÉáê=Ççã~áåë=~êÉ=ìëì~ääó=ÅçååÉÅíÉÇ=Äó=ÑäÉñáÄäÉ=äáåâÉê=êÉÖáçåëI=ïÜáÅÜ=~êÉ=éêçåÉ=íç=éêçíÉçäóëáëK=qÜáë=ÅÜ~ê~ÅíÉêáëíáÅ= áë=ìíáäáòÉÇ=

áå= íÜÉ= ÄáçÅÜÉãáÅ~ä= íÉÅÜåáèìÉ= çÑ= äáãáíÉÇ= éêçíÉçäóëáëI= ïÜáÅÜ= ïáää= ÄÉ=ÇáëÅìëëÉÇ=ä~íÉêK===

=========================================S=ÜííéWLLí~êÖÉíÇÄKéÇÄKçêÖLëí~íáëíáÅëLq~êÖÉípí~íáëíáÅëKÜíãä=

= OR

Alternative Expression Systems and Refolding qÜÉêÉ=Ü~îÉ=ÄÉÉå=ÉÑÑçêíë= íç= íêó= íç=ÇÉíÉêãáåÉ=ïÜáÅÜ=éêçíÉáå=éêçéÉêíáÉë=

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==Figure 5 Schematic illustration of the filtration dot blot (FiDo) procedure. Cells are

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protein is analyzed by dot blots.

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= PP

Äìí= ÄÉåÉÑáÅá~ä= ãìí~íáçåë= ~êÉ= åÉÉÇÉÇK= fÑ= íÜÉ= éêçíÉáå= Ü~ë= ~å= É~ëáäó=~ëë~óÉÇ= ÑìåÅíáçåI= íÜáë= ÅçìäÇ= áå= éêáåÅáé~ä= ÄÉ= ìëÉÇ= íç= ëÅêÉÉå= Ñçê=áåÅêÉ~ëÉÇ= ÉñéêÉëëáçå= çê= áåÅêÉ~ëÉÇ= Éåòóã~íáÅ= ~Åíáîáíó= xNMTzK= qÜáë=

ëíê~íÉÖó=Ü~ë=~=äáãáíÉÇ=íÜêçìÖÜéìí=ëáåÅÉ=ãçëí=~ëë~óë=~êÉ=“éä~íÉJÄ~ëÉÇÒ=~åÇ= ~= ä~êÖÉ= ã~àçêáíó= çÑ= ~ää= éêçíÉáåë= Çç= åçí= Ü~îÉ= ~å= É~ëó= ÜáÖÜJíÜêçìÖÜéìí= ~ëë~óK= qÜÉ= ãÉíÜçÇë= ãÉåíáçåÉÇ= É~êäáÉê= Ñçê= ëÅêÉÉåáåÖ=

ÉñéêÉëëáçå= é~ê~ãÉíÉêë= EÉÖK= VSJïÉää= Ä~ëÉÇ= Ñáäíê~íáçå= çê= éìêáÑáÅ~íáçå=~ëë~óëF=ÅçìäÇ=~äëç=ÄÉ=ìëÉÇ=íç=ëÅêÉÉå=Ñçê=ëí~Äáäáíó=çê=ëçäìÄáäáíóI=~äíÜçìÖÜ=

íÜÉ= íÜêçìÖÜéìí=~åÇ= íÜÉ=åìãÄÉê=çÑ=ÅçåëíêìÅíë= íÜ~í=ÅçìäÇ=ÄÉ= ëÅêÉÉåÉÇ=ïçìäÇ=ÄÉ=äáãáíÉÇK==fåëíÉ~Ç= ëÉîÉê~ä=ãÉíÜçÇë= íÜ~í= ëÅêÉÉå= Ñçê= ëçäìÄäÉ=éêçíÉáå=ÉñéêÉëëáçå=~í=

íÜÉ=Åçäçåó=äÉîÉä=Ü~îÉ=ÄÉÉå=ÇÉîÉäçéÉÇK===

Colony Screening Methods Methods Based on Reporter Proteins qç= Ç~íÉI= íÜÉ= ãçëí= ìëÉÇ= ëíê~íÉÖó= íç= ëÅêÉÉå= Ñçê= ëçäìÄäÉ= éêçíÉáå=ÉñéêÉëëáçå= áå= bK= Åçäá= áë= íÜÉ= Ñìëáçå= çÑ= ~= êÉéçêíÉê= éêçíÉáå= íç= íÜÉ= `J

íÉêãáåìë=çÑ=íÜÉ=éêçíÉáå=çÑ=áåíÉêÉëíK=qÜÉ=áÇÉ~=áë=~ë=ÑçääçïëW=fÑ=íÜÉ=í~êÖÉí=éêçíÉáå= áë=ÅçêêÉÅíäó=ÑçäÇÉÇ=íÜÉ=êÉéçêíÉê=éêçíÉáå=ïáää=~äëç=Ü~îÉ=íáãÉ=íç=ÑçäÇ= ÅçêêÉÅíäóK=eçïÉîÉê= íÜÉ= êÉîÉêëÉ= áë= ~äëç= íêìÉK= fÑ= íÜÉ= í~êÖÉí=éêçíÉáå=

ÇçÉë= åçí= ÑçäÇ= ÅçêêÉÅíäó= ~åÇ= ~ÖÖêÉÖ~íÉëI= íÜáë= ïáää= áåíÉêÑÉêÉ= ïáää= íÜÉ=ÑçäÇáåÖ= çÑ= íÜÉ= êÉéçêíÉê= éêçíÉáåI= ïÜáÅÜ= ëìÄëÉèìÉåíäó= ÇçÉë= åçí= ÑçäÇ=ÅçêêÉÅíäó= EcáÖìêÉ= SFK= _ó=ãçåáíçêáåÖ= íÜÉ= êÉéçêíÉê= éêçíÉáåI= íÜÉ= ÑçäÇáåÖ=

óáÉäÇ=çê=“ëçäìÄáäáíóÒ=çÑ=íÜÉ=í~êÖÉí=éêçíÉáå=Å~å=ÄÉ=Éëíáã~íÉÇK==qÜÉêÉ=~êÉ=ëÉîÉê~ä= êÉéçêíÉê=éêçíÉáåë= íÜ~í=Ü~îÉ=ÄÉÉå=ìëÉÇ=íç=ëÅêÉÉå= Ñçê=

ëçäìÄäÉ= éêçíÉáå= ÉñéêÉëëáçå= ~í= íÜÉ= Åçäçåó= äÉîÉäK= =j~ñïÉää= Éí= ~äK= Ü~îÉ=ÇÉëÅêáÄÉÇ= ~= ãÉíÜçÇ= Ä~ëÉÇ= çå= íÜÉ= Ñìëáçå= çÑ= ÅÜäçê~ãéÜÉåáÅçä=~ÅÉíóäíê~åëÑÉê~ëÉ=E`^qF=íç=íÜÉ=éêçíÉáå=çÑ=áåíÉêÉëí=xNMUzK=páåÅÉ=íÜÉ=`^q=

éêçíÉáå= ã~âÉë= íÜÉ= Üçëí= ÅÉääë= êÉëáëí~åí= íç= íÜÉ= ~åíáÄáçíáÅ=ÅÜäçê~ãéÜÉåáÅçäI=ëçäìÄáäáíó=ÅçìäÇ=ÄÉ=ãçåáíçêÉÇ=Äó=íÜÉ=ÖêçïíÜ=çÑ=ÅÉääë=

çå= éä~íÉë= Åçåí~áåáåÖ= íÜÉ= ~åíáÄáçíáÅK= `Éääë= ÉñéêÉëëáåÖ= éêçíÉáå= ~ë=áåÅäìëáçå=ÄçÇáÉë=ÉñÜáÄáíÉÇ=ÇÉÅêÉ~ëÉÇ=~åíáÄáçíáÅ=êÉëáëí~åÅÉ=Åçãé~êÉÇ=íç=

ÅÉääë= ÉñéêÉëëáåÖ= ëçäìÄäÉ= éêçíÉáåëK= ^êåçäÇ= ~åÇ= ÅçJïçêâÉêë= ìëÉÇ= íÜÉ=

= PQ

`^q= ãÉíÜçÇ= íç= ëÅêÉÉå= Ñçê= ëçäìÄáäáëÉÇ= ~åÇ= ÉîçäîÉÇ= mQRM= î~êá~åíë=xNMVzK==

=

==Figure 6 Schematic illustration of the principle behind folding reporters. (A) If the

target proteins folds correctly the reporter will also reach its native conformation and the activity / function can be measured. (B) A target proteins that misfolds will

interfere with the folding of the reporter which will not exhibit its function.

=qÜÉ=ìëÉ=çÑ=~å=~åíáÄáçíáÅ=ã~êâÉê=Ü~ë=íÜÉ=éçíÉåíá~ä=íç=ëÅêÉÉå=îÉêó=ä~êÖÉ=

äáÄê~êáÉë= ëáåÅÉ= çåäó= íÜÉ= ÅÉää= Åçåí~áåáåÖ= éä~ëãáÇë= ÅçÇáåÖ= Ñçê= ëçäìÄäÉ=î~êá~åíë=ïáää=ëìêîáîÉ=ïÜÉå=éä~íÉÇ=çå=~åíáÄáçíáÅëK==

qÜÉ=ÑçäÇáåÖ=ã~êâÉê=íÜ~í=Ü~ë=~ííê~ÅíÉÇ=íÜÉ=ãçëí=~ííÉåíáçå=íç=Ç~íÉ=ï~ë=ÇÉëÅêáÄÉÇ=Äó=t~äÇç=Éí=~äK=áå=NVVV=xNNMzK=qÜÉ=ãÉíÜçÇ=áë=Ä~ëÉÇ=çå=íÜÉ=Ñìëáçå=çÑ=íÜÉ=í~êÖÉí=éêçíÉáå=íç=íÜÉ=kJíÉêãáåìë=çÑ=íÜÉ=ÖêÉÉå=ÑäìçêÉëÅÉåí=

éêçíÉáå=EdcmFK=tÜÉå=íÜÉ= í~êÖÉí=éêçíÉáå= áë=ÉñéêÉëëÉÇ= áå=~=ëçäìÄäÉ=~åÇ=ëí~ÄäÉ=ÑçêãI=dcm=ïáää=ÑçäÇ=ÅçêêÉÅíäó=~åÇ=ÑäìçêÉëÅÉåÅÉ=Å~å=ÄÉ=ÇÉíÉÅíÉÇK=

lå=íÜÉ=çíÜÉê=Ü~åÇ=áÑ=íÜÉ=í~êÖÉí=áë=ÉñéêÉëëÉÇ=áåíç=áåÅäìëáçå=ÄçÇáÉëI=dcm=ïáää=åçí=Ü~îÉ= íáãÉ= íç= ÑçäÇ=~åÇ=åç= ÑäìçêÉëÅÉåÅÉ=Å~å=ÄÉ=ÇÉíÉÅíÉÇK=qÜáë=ãÉíÜçÇ=Ü~ë=ÄÉÉå=ìëÉÇ= áå=ÄçíÜ= äáèìáÇ=ÅìäíìêÉ=~åÇ=~í= íÜÉ=Åçäçåó= äÉîÉäK=

cçê=éêçíÉáå=ÉñéêÉëëáçå=áå=äáèìáÇ=ÅìäíìêÉI=t~äÇç=Éí=~äK=êÉéçêíÉÇ=~=îÉêó=ÖççÇ=ÅçêêÉä~íáçå=ÄÉíïÉÉå= ÑäìçêÉëÅÉåÅÉ=~åÇ= íÜÉ= ê~íáç=ÄÉíïÉÉå= ëçäìÄäÉ=

îÉêëìë=áåëçäìÄäÉ=éêçíÉáå=xNNMzK==qÜÉ=dcm=ãÉíÜçÇ=Ü~ë=~äëç=ÄÉÉå=ìëÉÇ=áå=åìãÉêçìë=Å~ëÉë=íç=ëÉêîÉ=~ë=êÉéçêíÉê=áå=áå=îáíêç=Éîçäìíáçå=ÉñéÉêáãÉåíëK=

bñ~ãéäÉë=ïÜÉêÉ= íÜÉ=ãÉíÜçÇ=Ü~ë=ÄÉÉå=ìëÉÇ= íç=çÄí~áå= ëçäìÄäÉ=éêçíÉáå=ÉñéêÉëëáçå= Ñçê= ëíêìÅíìê~ä= ëíìÇáÉë= Ü~îÉ= ÄÉÉå= ÇÉëÅêáÄÉÇ= Äó=t~äÇç= ~åÇ=

ÅçJïçêâÉêë= ~åÇ=_çïáÉ= ~åÇ= ÅçJïçêâÉêëK=t~äÇç= ~åÇ= ÅçJïçêâÉêë= ìëÉÇ=íÜÉ= ãÉíÜçÇ= íç= ÉîçäîÉ= ëçäìÄäÉ= î~êá~åíë= çÑ= ~= éêçíÉáå= Ñêçã= ~=

= PR

ÜóéÉêíÜÉêãçéÜáäáÅ= çêÖ~åáëã= íÜ~í= ï~ë= áåëçäìÄäÉ= ~ë= ~= tq= ÅçåëíêìÅí=xVUzK= _çïáÉ= ~åÇ= ÅçJïçêâÉêë= ìëÉÇ= ê~åÇçã= ãìí~ÖÉåÉëáë= ÑçääçïÉÇ= Äó=dcm=ëÅêÉÉåáåÖ=íç=ÉîçäîÉ=ãçêÉ=ëçäìÄäÉ=î~êá~åíë=çÑ=~=p^jLqbi=Ççã~áå=

xNNNzK= bñéêÉëëáçå= ëÅêÉÉåáåÖ=ìëáåÖ=dcm= áå= ÅçãÄáå~íáçå=ïáíÜ= ÇáêÉÅíÉÇ=Éîçäìíáçå= Ü~ë= ~äëç= ÄÉÉå= ìëÉÇ= íç= áåÅêÉ~ëÉ= íÜÉ= ÉñéêÉëëáçå= çÑ=ÄáçíÉÅÜåçäçÖáÅ~ääó= áãéçêí~åí= éêçíÉáåëK= s~å=aÉå=_ÉêÖ= Éí= ~äK= ìëÉÇ= íÜÉ=

ãÉíÜçÇ=íç=áåÅêÉ~ëÉ=íÜÉ=óáÉäÇ=çÑ=êÉÅçãÄáå~åíäó=ÉñéêÉëëÉÇ=qbs=éêçíÉ~ëÉ=xNMRzK==

qÜÉ=åÉÖ~íáîÉ=~ëéÉÅí=çÑ=ìëáåÖ=Ñìëáçå=éêçíÉáå=Ä~ëÉÇ=ãÉíÜçÇë=áë=íÜ~í=íÜÉ=êÉéçêíÉê=éêçíÉáå=ãáÖÜí=~ÑÑÉÅí=íÜÉ=ëçäìÄáäáíó=çÑ=íÜÉ=í~êÖÉí=éêçíÉáåK=qÜÉêÉ=~êÉ= Éñ~ãéäÉë= ïÜÉêÉ= áí= Ü~ë= ÄÉÉå= ëÜçïå= íÜ~í= dcm= ã~âÉë= íÜÉ= í~êÖÉí=

éêçíÉáå= äÉëë= ëçäìÄäÉ= xSSzK= qÜÉêÉ= áë= ~äëç= ~= êáëâI= ïÜÉå= áåíêçÇìÅáåÖ=ê~åÇçã=ãìí~íáçåëI=íÜ~í=~=o_p=ïáää=ÄÉ=ÅêÉ~íÉÇ=àìëí=ÄÉÑçêÉ=íÜÉ=êÉéçêíÉê=éêçíÉáåI= êÉëìäíáåÖ= áå= çåäó= ÉñéêÉëëáçå= çÑ= íÜÉ= êÉéçêíÉê= éêçíÉáå= xNMQzK=

^åçíÜÉê=Çê~ïÄ~Åâ=çÑ=ìëáåÖ=~=ä~êÖÉ=êÉéçêíÉê=éêçíÉáå=Ñìëáçå=áë=íÜ~í=íÜÉó=Ü~îÉ= íç= ÄÉ= êÉãçîÉÇ= ÄÉÑçêÉ= íÜÉ= éêçíÉáå= çÑ= áåíÉêÉëí= Å~å= ÄÉ= ìëÉÇ= Ñçê=

ëíêìÅíìê~ä=ëíìÇáÉëK==

Peptide Based Reporters qç= ÅáêÅìãîÉåí= íÜÉ= éêçÄäÉãë= ~ëëçÅá~íÉÇ= ïáíÜ= ~= ä~êÖÉ= ëçäìÄáäáíó=

êÉéçêíÉêI=ãÉíÜçÇë=Ü~îÉ=ÄÉÉå=ÇÉîÉäçéÉÇ=ìëáåÖ=~=ëã~ääÉê=éÉéíáÇÉ=ÑìëÉÇ=íç=íÜÉ=éêçíÉáå=çÑ=áåíÉêÉëíK=qÜÉëÉ=ãÉíÜçÇë=~êÉ=ìëì~ääó=Ä~ëÉÇ=çå=ëéäáííáåÖ=

~=êÉéçêíÉê=éêçíÉáå=áåíç=íïç=éáÉÅÉëK=låÉ=é~êí=çÑ=íÜÉ=êÉéçêíÉê=éêçíÉáå=áë=ÑìëÉÇ=íç=íÜÉ=í~êÖÉí=ïÜáäÉ=íÜÉ=çíÜÉê=é~êí= áë=ÉñéêÉëëÉÇ=~äçåÉ=EcáÖìêÉ=TFK=qÜÉ=íïç=éêçíÉáå=éáÉÅÉë=~êÉ=åçí=~ÅíáîÉ=çå=íÜÉáê=çïå=Äìí=ïÜÉå=íÜÉó=~êÉ=

“ìåáíÉÇÒ=íÜÉ=çêáÖáå~ä=éêçíÉáå=ÑìåÅíáçå=áë=êÉëíçêÉÇK=fÑ=íÜÉ=éêçíÉáå=ÑìëÉÇ=íç= íÜÉ= ëéäáí= í~Ö= áë= áåëçäìÄäÉI= åç= ÅçãéäÉãÉåí~íáçå=ïáää= çÅÅìêK=lå= íÜÉ=çíÜÉê=Ü~åÇI=áÑ=íÜÉ=éêçíÉáå=áë=ëçäìÄäÉI=íÜÉ=íïç=éêçíÉáå=éáÉÅÉë=ïáää=ÄÉ=~ÄäÉ=

íç=ÅçãéäÉãÉåí=É~ÅÜ=çíÜÉê=~åÇ=íÜÉ=êÉéçêíÉê=ïáää=ÄÉ=~ÅíáîÉK===táÖäÉó=Éí=~äK=Ü~îÉ=ÇÉëÅêáÄÉÇ=~=ãÉíÜçÇ=ïÜÉêÉ=íÜÉ=ÄÉí~JÖ~ä~ÅíçëáÇ~ëÉ=EβJÖ~äF= éêçíÉáå= ï~ë= ëéäáí= xNNOzK= qÜÉ= éêçíÉáå= ï~ë= ëéäáí= áåíç= ~= úRM= ~~=Ñê~ÖãÉåíI= ïÜáÅÜ= ï~ë= ÑìëÉÇ= íç= íÜÉ= éêçíÉáå= çÑ= áåíÉêÉëíI= ~åÇ= íÜÉ=êÉã~áåáåÖ=NPR=âa~=é~êí=çÑ=βJÖ~ä=ï~ë=ÅçJÉñéêÉëëÉÇK=qÜÉ=~ìíÜçêë=ÑçìåÇ=~= ÖççÇ= ÅçêêÉä~íáçå= ÄÉíïÉÉå= βJÖ~ä= ~Åíáîáíó= ~åÇ= ÑçäÇáåÖ= óáÉäÇK=

= PS

råÑçêíìå~íÉäó=áí=Ü~ë=ÄÉÉå=ëÜçïå=íÜ~í=íÜÉ=Ñìëáçå=çÑ=íÜÉ=úRM=~~=éÉéíáÇÉ=íç=ëçäìÄäÉ=éêçíÉáåë=Å~å=êÉåÇÉê=íÜÉã=áåëçäìÄäÉ=xNNPzK===

=

==Figure 7 Illustration of the principle behind split protein based solubility screens. The

reporter protein is split into two pieces and one part is fused to the target protein. (A) The target protein is expressed in an non-aggregated from and the fused reporter peptide can complement the other part of the reporter protein which will become

functional (B) The protein is produced in an aggregated form and the fused reporter peptide will not be able to complement the other part of the reporter protein and the

activity will not be restored.

=

oÉÅÉåíäó=~=éêçãáëáåÖ=ãÉíÜçÇ=ìíáäáòáåÖ=~=ëéäáí=dcm=Ü~ë=ÄÉÉå=ÇÉëÅêáÄÉÇ=xNNQzK=få=íÜáë=ëíìÇó=íÜÉ=ëã~ää=ëéäáí=éÉéíáÇÉ=ï~ë=çéíáãáòÉÇ=íç=åçí=~ÑÑÉÅí=íÜÉ= ëçäìÄáäáíó= çÑ= íÜÉ= í~êÖÉí= éêçíÉáåK= aÉéÉåÇáåÖ= çå=ïÜÉíÜÉê= íÜÉ= dcm=

éêçíÉáå= Eä~ÅâáåÖ= íÜÉ= ä~ëí= ÄÉí~= ëíê~åÇF=ï~ë= ÅçJáåÇìÅÉÇ=çê= ëÉèìÉåíá~ääó=áåÇìÅÉÇI=ÉáíÜÉê=íçí~ä=çê=ëçäìÄäÉ=éêçíÉáå=ÉñéêÉëëáçåI=êÉëéÉÅíáîÉäóI=ÅçìäÇ=ÄÉ= ÇÉíÉÅíÉÇK= qÜÉ= ëéäáí= dcm= ~ééêç~ÅÜ= Ü~ë= ÄÉÉå= ëìÅÅÉëëÑìääó= ìëÉÇ= áå=

ÅçãÄáå~íáçå=ïáíÜ=ê~åÇçã=ãìí~ÖÉåÉëáë=íç=áåÅêÉ~ëÉ=íÜÉ=äÉîÉä=çÑ=ëçäìÄäÉ=éêçíÉáå= ÉñéêÉëëáçå= Ñçê= ëÉîÉê~ä= êÉÅ~äÅáíê~åí= éêçíÉáåë= xNMQzK= låÉ=

éçíÉåíá~ä= Çê~ïÄ~Åâ=ïáíÜ= íÜÉ= ëéäáí=dcm=ãÉíÜçÇ= áë= íÜ~í= íïç= éä~ëãáÇë=Ü~îÉ= íç= ÄÉ= ìëÉÇ= ÇìêáåÖ= íÜÉ= ÉñéêÉëëáçå= ÉñéÉêáãÉåíI= çåÉ= Å~êêóáåÖ= íÜÉ=í~êÖÉí=éêçíÉáå=~åÇ=çåÉ=Åçåí~áåáåÖ=íÜÉ=dcmK=

=

= PT

mÉ~ÄçÇó=~åÇ=^äJ_áí~ê=Ü~îÉ=ÇÉîÉäçéÉÇ=~=ãÉíÜçÇ=íÜ~í=ÇçÉë=åçí=êÉèìáêÉ=íÜÉ= Ñìëáçå= çÑ= ~= éêçíÉáå= çê= éÉéíáÇÉ= xNNRzK= fåëíÉ~Ç= ëçäìÄäÉ= éêçíÉáå=ÉñéêÉëëáçå=áë=ÇÉíÉÅíÉÇ=Äó=ÅçîÉêáåÖ=íÜÉ=ÅçäçåáÉë=ïáíÜ=~=ä~óÉê=çÑ=~Ö~êçëÉK=

qÜÉ=ÅÉääë= äóëÉ=~åÇ=íÜÉ=éêçíÉáåë=~êÉ=êÉäÉ~ëÉÇ=Ñêçã=íÜÉ=ÅÉääë=~åÇ=ÇáÑÑìëÉ=íÜêçìÖÜ=íÜÉ=~Ö~êçëÉ=ä~óÉêK=i~êÖÉ=~ÖÖêÉÖ~íÉëI=ëìÅÜ=~ë=áåÅäìëáçå=ÄçÇáÉëI=ÇáÑÑìëÉ= îÉêó= ëäçïäó= íÜêçìÖÜ= íÜÉ= ~Ö~êçëÉ= ä~óÉêI=ïÜáäÉ= ëçäìÄäÉ=éêçíÉáåë=

ÇáÑÑìëÉ=ãìÅÜ=Ñ~ëíÉêK=_ó=éä~ÅáåÖ=~=åáíêçJÅÉääìäçëÉ=ãÉãÄê~åÉ=çå=íçé=çÑ=íÜÉ= ~Ö~êçëÉ= ä~óÉêI= íÜÉ= éêçíÉáå= Å~å= ÄÉ= Å~éíìêÉÇK= qÜÉ= ãÉãÄê~åÉ= Å~å=

íÜÉå= ÄÉ= éêçÄÉÇ= ìëáåÖ= ëí~åÇ~êÇ= áããìåçÅÜÉãáÅ~ä= íÉÅÜåáèìÉëK= qÜÉ=Çáë~Çî~åí~ÖÉ=çÑ=ìëáåÖ=íÜáë=ãÉíÜçÇ=áë=íÜ~í=~=äáãáíÉÇ=åìãÄÉê=çÑ=ÅçäçåáÉë=Å~å=ÄÉ=ëÅêÉÉåÉÇ=ëáåÅÉ=íÜÉ=ÇáÑÑìëáçå=çÑ=íÜÉ=éêçíÉáåë=“ÄäìêëÒ=íÜÉ=áã~ÖÉK==

=

= PU

The Colony Filtration Blot (Paper II) tÉ= Ü~îÉ= éêÉîáçìëäó= ëÜçïå= íÜ~í= íÜÉ= ìëÉ= çÑ= Ñáäíê~íáçå= Å~å= ëÉêîÉ= ~ë= ~=ãÉíÜçÇ=íç=ëÉé~ê~íÉ=ëçäìÄäÉ=éêçíÉáå=Ñêçã=áåÅäìëáçå=ÄçÇáÉë=xVPzK=aìÉ=íç=

íÜÉ= ëìÅÅÉëë= çÑ= íÜáë= ãÉíÜçÇI= ïÉ= ÉñéäçêÉÇ= áÑ= Ñáäíê~íáçå= ÅçìäÇ= ÄÉ=éÉêÑçêãÉÇ= ~í= íÜÉ= Åçäçåó= äÉîÉäK= tÉ= ï~åíÉÇ= íç= ìëÉ= ~= aìê~éçêÉ=

ãÉãÄê~åÉ=ëáåÅÉ= áí= áë= íÜÉ=ë~ãÉ=íóéÉ=çÑ=ãÉãÄê~åÉ=éêÉëÉåí= áå=íÜÉ=ÑáäíÉê=éä~íÉë= ìëÉÇ= áå= íÜÉ= VS= ïÉää= ëÅêÉÉåK= qÜÉ= Ñáêëí= ëíÉé= ï~ë= íç= áåîÉëíáÖ~íÉ=

ïÜÉíÜÉê= íÜÉ= ÅçäçåáÉë= ÅçìäÇ= ÄÉ= “éÉÉäÉÇÒ= çÑÑ= ~= i_= éä~íÉ= ìëáåÖ= íÜÉ=aìê~éçêÉ=ãÉãÄê~åÉI=~åÇ=áåÇÉÉÇI=íÜÉ=ÅçäçåáÉë=ëíìÅâ=íç=íÜÉ=ãÉãÄê~åÉK=

qÜÉ=åÉñí=ï~ë=íç=éÉêÑçêã=íÜÉ=äóëáë=~åÇ=Ñáäíê~íáçå=ëíÉéK=tÉ=ÄÉäáÉîÉÇ=íÜ~í=ëçãÉ=âáåÇ=çÑ=ÑçêÅÉ=ï~ë=åÉÉÇÉÇ=íç=ãçîÉ=íÜÉ=éêçíÉáå=íÜêçìÖÜ=íÜÉ=ÑáäíÉê=

ãÉãÄê~åÉK= pÉîÉê~ä= ÇáÑÑÉêÉåí= ëÉíJìéë= ïÉêÉ= íêáÉÇW= íê~åëÑÉêJë~åÇïáÅÜÉë=êÉëÉãÄäáåÖ=kçêíÜÉêåJ_äçíë=EcáÖìêÉ=U=~FI=ìëáåÖ=î~Åììã=EcáÖìêÉ=U=ÄFI=çê=~=ëáãéäÉ=ÑáäíÉê=ë~åÇïáÅÜ=EcáÖìêÉ=U=ÅF==

==

=

DuraporeNitrocellulose

Vacuum

Funnel

3 MM

DuraporeNitrocellulose

3 MM

3 MM

LysisBuffer

DuraporeNitrocellulose

3 MM

Capi

llary

forc

e

a b c

==Figure 8 Early prototypes of the colony filtration step. (A) A setup resembling a

Northern blot transfer sandwich. The capillary force drives the transfer of proteins

from the colonies through the Durapore membrane onto the nitrocellulose membrane (B) The filter sandwich placed in a Büchner funnel. By applying vacuum the proteins

are forced through the Durapore membrane. (C) The filter sandwich was placed in a Petri dish and freeze thawed. This was the most successful experimental setup.

=

= PV

tÉ= ÑçìåÇ= íÜ~í= íÜÉ= ãçëí= ÉÑÑáÅáÉåí= Åçäçåó= Ñáäíê~íáçå= ãÉíÜçÇ= ï~ë= íç=ÑêÉÉòÉJíÜ~ï= ~= ÑáäíÉê= ë~åÇïáÅÜ= ÅçåëáëíáåÖ= çÑ= ~=aìê~éçêÉ=ãÉãÄê~åÉ= ~=åáíêçÅÉääìäçëÉ=ãÉãÄê~åÉ=~åÇ=~=tÜ~íã~å=Pjj=é~éÉê=ëç~âÉÇ=áå=äóëáë=

ÄìÑÑÉêK=qÜÉ=åáíêçÅÉääìäçëÉ=Å~éíìêÉë=íÜÉ=éêçíÉáå=êÉäÉ~ëÉÇ=Ñêçã=íÜÉ=äóëÉÇ=ÅÉääë=~åÇ=Å~å=ëìÄëÉèìÉåíäó=ÄÉ=éêçÄÉÇ=ìëáåÖ=ëí~åÇ~êÇ=áããìåçÅÜÉãáÅ~ä=ãÉíÜçÇë= EcáÖìêÉ=VFK=tÉ= ä~íÉê= êÉ~äáòÉÇ= íÜ~í= ëçãÉ=çÑ= íÜÉ= ÉñéÉêáãÉåí~ä=

ëÉíìéë=çÑ=íÜÉ=`çcá=Ääçí=ãÉíÜçÇ=ëçãÉïÜ~í=êÉëÉãÄäÉÇ=~=ãÉíÜçÇ=ìëÉÇ=íç=ëÅêÉÉå=Ñçê=~åíáÄçÇó=Ñê~ÖãÉåíë=ÉñéêÉëëÉÇ=áå=Ä~ÅíÉêá~=xNNSzK==

=qÜÉ= Éñ~Åí=ãÉÅÜ~åáëã= íÜ~í= ÇêáîÉë= íÜÉ= éêçíÉáå= íÜêçìÖÜ= íÜÉ= aìê~éçêÉ=ãÉãÄê~åÉ= áë= åçí= ÅäÉ~êI= Äìí=ãçëí= äáâÉäó= áí= áë= “ëáãéäÉÒ= ÇáÑÑìëáçå= íÜ~í=

~ÅÅçìåíë= Ñçê= íÜÉ= éêçíÉáå= ãçîÉãÉåíK= páåÅÉ= íÜÉ= ãÉíÜçÇ= áë= Ä~ëÉÇ= çå=Ñáäíê~íáçå= ~í= íÜÉ= Åçäçåó= äÉîÉäI= ÑçääçïÉÇ= Äó= ~= ÄäçííáåÖ= éêçÅÉÇìêÉI= ïÉ=ÅÜççëÉ=íÜÉ=Å~ää=íÜÉ=ãÉíÜçÇ=`çäçåó=cáäíê~íáçå=Ääçí=çê=`çcá=ÄäçíK==

=

==Figure 9 Schematic illustration of the CoFi blot procedure. (A) After the expression

has been induced, the filter sandwich is made. (B) The cells are lysed and soluble proteins diffuse through the filter membrane while inclusion bodies are retained. (C)

The nitrocellulose membrane is probed with standard immunochemicals. (D) The first CoFi blot. Colonies expressing soluble proteins were plated as a plus sign surrounded by colonies expressing a protein in inclusion bodies. Only the soluble protein is

visible on the blot, seen as a plus sign.

=

= QM

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éêçíÉáåë=áå=íïç=ÇáÑÑÉêÉåí=ÉñéêÉëëáçå=îÉÅíçêë=EkJíÉêãáå~ä=cä~Ö=Ó=çê=eáëJí~ÖFI= Ñçê=~= íçí~ä=çÑ=NMU=ìåáèìÉ=ÅçåëíêìÅíëI=ï~ë=ìëÉÇK= =qÜÉ=ÅçåëíêìÅíë=ïÉêÉ=ÄçíÜ=Öêçïå=~ë=ÅçäçåáÉë=~åÇ=áå=äáèìáÇ=ÅìäíìêÉK=qÜÉ=ÅçåëíêìÅíë=íÜ~í=

ïÉêÉ=Öêçïå=~åÇ= áåÇìÅÉÇ=~ë=ÅçäçåáÉë=ïÉêÉ=ëìÄàÉÅíÉÇ=íç=íÜÉ=`çcá=Ääçí=ãÉíÜçÇ=ïÜáäÉ= íÜÉ= ÅÉääë=Öêçïå= áå= äáèìáÇ=ÅìäíìêÉ=ïÉêÉ=Ü~êîÉëíÉÇI= äóëÉÇ=

~åÇ= ÅÉåíêáÑìÖÉÇK= qÜÉ= íçí~ä= éêçíÉáå= Ñê~Åíáçå= EëçäìÄäÉ= ~åÇ= áåëçäìÄäÉ=éêçíÉáåëF=~åÇ=íÜÉ=ëçäìÄäÉ=Ñê~Åíáçå=ïÉêÉ=Ççí=ÄäçííÉÇ=EcáÖìêÉ=NMFK=tÜÉå=Åçãé~êáåÖ=íÜÉ=íïç=ãÉíÜçÇë=íÜÉêÉ=áë=~å=UQ=B=çîÉê~ää=~ÖêÉÉãÉåíK=qÜÉêÉ=

áë=~äëç=~=Ñ~áêäó=ÖççÇ=ÅçêêÉä~íáçå=ÄÉíïÉÉå=íÜÉ=ÉñéêÉëëáçå=äÉîÉä=ÇÉíÉÅíÉÇ=~í=íÜÉ=Åçäçåó=äÉîÉä=ìëáåÖ=íÜÉ=`çcá=Ääçí=ãÉíÜçÇ=~åÇ=íÜÉ=ÉñéêÉëëáçå=äÉîÉä=áå=äáèìáÇ=ÅìäíìêÉK=Em~éÉê=ffF=

=Figure 10 The CoFi blot method compared to centrifugation. Constructs from 32

different proteins in two different expression vectors were used to validate the CoFi blot. Cells were grown both as colonies and in liquid culture. The colonies were subjected to the CoFi blot method while for the cells grown in liquid culture, soluble

protein was separated from inclusion bodies by centrifugation. Dot blots were made of both the soluble fraction and the total protein fraction (soluble and inclusion

bodies). Constructs with no total protein expression were excluded from the statistics, indicated by a zero.

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In vitro Evolution and the CoFi blot

(Paper III) ^ë= åçíÉÇ= É~êäáÉêI= íÜÉ= êÉÅçãÄáå~åí= çîÉêÉñéêÉëëáçå= çÑ= Éìâ~êóçíáÅ=éêçíÉáåë= ~êÉ= ãìÅÜ= ãçêÉ= ÅÜ~ääÉåÖáåÖ= íÜ~å= íÜÉ= çîÉêÉñéêÉëëáçå= çÑ=éêçâ~êóçíáÅ= éêçíÉáåëK= qÜáë= Ü~ë= ÄÉÉå= ~ííêáÄìíÉÇ= íç= ëÉîÉê~ä= ÇáÑÑÉêÉåí=

Ñ~Åíçêë= ëìÅÜ= ~ë= íÜÉ= êÉèìáêÉãÉåí= çÑ= ëéÉÅáÑáÅ= ÅÜ~éÉêçåÉëI= íÜÉ= åÉÉÇ= Ñçê=éçëíJíê~åëä~íáçå~ä=ãçÇáÑáÅ~íáçåëI= ~åÇ=ÇáëìäÑáÇÉ= Ñçêã~íáçåK= páåÅÉ= íÜÉêÉ=áë=~=ÅçêêÉä~íáçå=ÄÉíïÉÉå=íÜÉ=ëáòÉ=çÑ=íÜÉ=éêçíÉáå=~åÇ=íÜÉ=ëìÅÅÉëë=ê~íÉ=çÑ=

ëçäìÄäÉ=çîÉêÉñéêÉëëáçå=áå=bK=ÅçäáI=çåÉ=~ééêç~ÅÜ=íÜ~í=Ü~ë=ÄÉÉå=ìëÉÑìä=áë=íÜÉ=ÅäçåáåÖ=~åÇ=ÉñéêÉëëáçå=çÑ=ëáåÖäÉ=Ççã~áåë=xSSzK=qÜÉ=äçÅ~íáçå=çÑ=íÜÉ=

Ççã~áå=ïáíÜáå=íÜÉ=éêçíÉáå=Å~å=ÉáíÜÉê=ÄÉ=ÉñéÉêáãÉåí~ääó=ÇÉíÉêãáåÉÇ=çê=Äó= ÄáçáåÑçêã~íáÅë= ãÉíÜçÇëK= qÜÉ= ÉñéÉêáãÉåí~ä= ~ééêç~ÅÜÉë= ~êÉ= Ä~ëÉÇ=çå= äáãáíÉÇ= éêçíÉçäóëáë= ~åÇ= ã~ëë= ëéÉÅíêçãÉíêó= EjpF= xNNTI= NNUzI==

ÇÉìíÉêáìã= ÉñÅÜ~åÖÉ= jp= EaujpF= xNNVz= çê= Äó= ÄáçáçåÑçêã~íáÅ=~ééêç~ÅÜÉë= xNOMJNOOzK= qÜÉ= ìëÉ= çÑ= äáãáíÉÇ= éêçíÉçäóëáë= çê=aujp= Ü~ë=

ÄÉÉå= ìëÉÇ= ëìÅÅÉëëÑìääó= íç= çÄí~áå= ÄÉííÉê= ÉñéêÉëëáåÖ= ÅçåëíêìÅíë= çê=ÅçåëíêìÅíë=ãçêÉ=Ñ~îçê~ÄäÉ=Ñçê=Åêóëí~ääáò~íáçåK=eçïÉîÉêI=íÜÉëÉ=ãÉíÜçÇë=

êÉèìáêÉ= íÜ~í= íÜÉ= Ñìää= äÉåÖíÜ= éêçíÉáå= Å~å= ÄÉ= ÉñéêÉëëÉÇ= ~åÇ= éìêáÑáÉÇK=^åçíÜÉê= éêçÄäÉã= áë= íÜ~í= ïÜÉå= íÜÉ= Ççã~áå= ÄçêÇÉêë= Ü~îÉ= ÄÉÉå=

áÇÉåíáÑáÉÇI=íÜÉ=ÅçåëíêìÅí=Ü~ë=íç=ÄÉ=êÉJÅäçåÉÇK===qÜÉêÉ= ~êÉ= ëÉîÉê~ä= ÇáÑÑÉêÉåí= ÄáçáåÑçêã~íáÅë= íççäë= íÜ~í= ~êÉ= ~î~áä~ÄäÉ= íç=

éêÉÇáÅí=Ççã~áå=ÄçêÇÉêëK=mêçÄ~Ääó=íÜÉ=ãçëí=ìëÉÇ=áë=íÜÉ=éêçíÉáå=Ñ~ãáäó=Ç~í~Ä~ëÉ=mc^jV= xNOMzK=qç=Ç~íÉI= íÜÉ=Ç~í~Ä~ëÉ=Åçåí~áåë=~äãçëí=UIMMM=

ìåáèìÉ= éêçíÉáå= Ñ~ãáäáÉëI= ïÜáÅÜ= Ü~îÉ= ~ää= ÄÉÉå= ã~åì~ääó= Åìê~íÉÇK= qç=éêÉÇáÅí=íÜÉ=Ççã~áå=Åçãéçëáíáçå=çÑ=íÜÉ=í~êÖÉí=éêçíÉáåI=íÜÉ=~ãáåç=~ÅáÇ=ëÉèìÉåÅÉ= áë= ëÉ~êÅÜÉÇ= ~Ö~áåëí= íÜÉ= Ç~í~Ä~ëÉ= ~åÇ= íÜÉ= éçëáíáçå= çÑ= íÜÉ=

ÄÉÖáååáåÖ= ~åÇ= íÜÉ= ÉåÇ=çÑ= íÜÉ= Ççã~áåë= ~êÉ= êÉéçêíÉÇK=eçïÉîÉêI= íÜÉëÉ=éêÉÇáÅíáçåë= Ü~îÉ= ~= ëáÖåáÑáÅ~åí= ÇÉÖêÉÉ= çÑ= ìåÅÉêí~áåíó= ~åÇ= ãìäíáéäÉ=

ÅçåëíêìÅíëI=ëí~êíáåÖ=~í=ÇáÑÑÉêÉåí=éçëáíáçåë=~êçìåÇ=íÜÉ=éêÉÇáÅíÉÇ=Ççã~áå=ÄçêÇÉêëI=~êÉ=íÜÉêÉÑçêÉ=åçêã~ääó=íÉëíÉÇ=Ñçê=ÉñéêÉëëáçå=EcáÖìêÉ=NNFK=qÜáë=

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F1 F2 F3

R1 R2

Amplify the gene with all primer combinations =F1 & R1, F1 & R2, F2 & R1, F2 & R2, F3 & R1 and F3 & R2 =

=Figure 11 A typical result from a PFAM database search of human multi-domain

protein. The domain cloning strategy is schematic outlined by the arrows, which indicate possible primer positions. All possible primer combinations are used to

increase the likelihood of creating a construct with soluble protein expression.

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Ä~ëÉÇ=çå=Ñê~ÖãÉåí~íáçå=çÑ=íÜÉ=loc=ÑçääçïÉÇ=Äó=ÉñéêÉëëáçå=ëÅêÉÉåáåÖ=xNMSI= NOPzK= få= ÄçíÜ= íÜÉëÉ= ÉÑÑçêíë= ëçäìÄäÉ= ÅçåëíêìÅíë= ~ééêçñáã~íÉäó=

ÅçêêÉëéçåÇáåÖ=íç=Ççã~áå=ÄçêÇÉêë=ïÉêÉ=çÄí~áåÉÇ=ÜçïÉîÉê=~=îÉêó=ä~êÖÉ=åìãÄÉê=çÑ=ÅäçåÉë=Ü~Ç=íç=ÄÉ=ëÅêÉÉåÉÇ=ëçãÉïÜ~í=äáãáíáåÖ=íÜÉ=éçíÉåíá~ä=çÑ=íÜÉ=ãÉíÜçÇëK=

=tÉ=Ü~îÉ=ÇÉîÉäçéÉÇ=~å=ÉñéÉêáãÉåí~ä=ëíê~íÉÖó=íÜ~í=ÇçÉë=åçí=êÉèìáêÉ=~åó=

éêáçê= âåçïäÉÇÖÉ= ~Äçìí= íÜÉ= Ççã~áå= Åçãéçëáíáçå= çÑ= íÜÉ= éêçíÉáåK= qÜÉ=ëíê~íÉÖó= êÉäáÉë= çå= ÅêÉ~íáåÖ= áåÅêÉãÉåí~ä= ÇÉäÉíáçåë= çÑ= íÜÉ= loc= Äó= ~=ãÉíÜçÇ= ÇÉëÅêáÄÉÇ= Äó= eÉåáâçÑÑ= xNOQz= EcáÖìêÉ= NOF= E`çããÉêÅá~ääó=

~î~áä~ÄäÉ= ~ë= íÜÉ= bê~ëÉJ^J_~ëÉ= ëóëíÉã= Ñêçã=mêçãÉÖ~FK=qÜÉ=ãÉíÜçÇ= áë=Ä~ëÉÇ=çå=íÜÉ=ëéÉÅáÑáÅ=ÇáÖÉëíáçå=çÑ=çåÉ=ëíê~åÇ=çÑ=íÜÉ=ak^=Eëëak^F=Äó=

bñçåìÅäÉ~ëÉ= fff= Ñêçã= ~= éêçíêìÇáåÖ= RÛ= çîÉêÜ~åÖ= ÖÉåÉê~íÉÇ= Äó=êÉëíêáÅíáçå=ÇáÖÉëíáçåK=qç=~ÅÜáÉîÉ= ~=ìåáÇáêÉÅíáçå~ä=ÇáÖÉëíáçåI= íÜÉ=çíÜÉê=“ÉåÇÒ=çÑ= íÜÉ=ak^= Ñê~ÖãÉåí= áë= éêçíÉÅíÉÇ=Äó= ~=PÛ= çîÉêÜ~åÖ=ïÜáÅÜ= áë=

åçí= ~ÅÅÉëëáÄäÉ= íç= íÜÉ= bñçåìÅäÉ~ëÉ= fffK= qç= ÅêÉ~íÉ= locë= çÑ= ÇáÑÑÉêÉåí=äÉåÖíÜëI=íáãÉÇ=~äáèìçíë=~êÉ=êÉãçîÉÇ=Ñêçã=íÜÉ=bñçåìÅäÉ~ëÉ=fff=êÉ~ÅíáçåK=oÉãçî~ä= çÑ= íÜÉ= ëëak^= áë= éÉêÑçêãÉÇ= Äó= pN= åìÅäÉ~ëÉK= qÜÉ= ak^=

Ñê~ÖãÉåí=áë=íÜÉå=êÉJäáÖ~íÉÇ=~åÇ=íê~åëÑçêãÉÇ=áåíç=~=ÅäçåáåÖ=ëíê~áåK==

= QQ

==

5'Overhang

3'Overhang

Exonuclease III

S1 nuclease

Ligase Ligase

Ligas

e

Timepoint 1 Timepoint 3Timepoint 2

Prepare DNA by restrictiondigestion with appropriate enzymes

Start deletion by addingExoIII and remove timedaliquotes

Remove ssDNA tailsusing S1 nuclease.

Restriction site leaving 5' overhangRestriction site leaving 3' overhangAtt-siteAffinity tagPromoter

6xHis-tag

ORF

1.

2.

3.

4.

5.

6.

7.

Religate plasmid

Transform library inte anE.coli cloning strain

==Figure 12 A schematic representation of the Erase-A-Base process. The plasmid is linearized by endonucelase digestion at two unique restriction sites introduced in the

cloning primer (2). The enzymes are chosen in such a way that one leaves a 3’ overhang protected from ExoIII digestion and the other leaves a 5’ overhang, which is

susceptible to the digestion. Samples from the ExoIII digestion are removed at timedintervals (3) and added to tubes containing S1 Nuclease, which removes the

remaining single-stranded tails. (4). The plasmid is re-ligated (5) and transformed into a E. coli cloning strain (6,7). The library is then recovered from the cloning strain

and transformed into an expression strain and screened for soluble expression with the CoFi blot.

=

tÉ= ëÉäÉÅíÉÇ=ON=Üìã~å=éêçíÉáåë=ïáíÜ=åç=çê=îÉêó= äçï= äÉîÉä=çÑ= ëçäìÄäÉ=éêçíÉáåë= ÉñéêÉëëáçå= Ñêçã= íÜÉ= ëÉí= íÜ~í=ï~ë= ìëÉÇ= íç= î~äáÇ~íÉ= íÜÉ=`çcá=Ääçí=ãÉíÜçÇ=EcáÖìêÉ=NPFK==

qÜÉ=éêçíÉáåë=ïÉêÉ=êÉJÅäçåÉÇ=áåíç=íÜÉ=ë~ãÉ=ÉñéêÉëëáçå=îÉÅíçê=íÜ~í=ï~ë=ìëÉÇ= Ñçê= íÜÉ= çêáÖáå~ä= ëÅêÉÉå= ïáíÜ= íÜÉ= ÉñÅÉéíáçå= íÜ~í= íïç= êÉëíêáÅíáçå=ëáíÉë= êÉèìáêÉÇ= áå= íÜÉ= ÇÉäÉíáçå= ãìí~ÖÉåÉëáë= éêçÅÉëë= ïÉêÉ= áåíêçÇìÅÉÇ=

ÄÉÑçêÉ=íÜÉ=locK=iáÄê~êáÉë= ÅçîÉêáåÖ= íÜÉ= ÉåíáêÉ= loc= ïÉêÉ= ÅêÉ~íÉÇI= É~ÅÜ= Åçåí~áåáåÖ= ~í=

äÉ~ëí=SIMMM=ìåáèìÉ=ÅäçåÉëK==^ééêçñáã~íÉäó=NIRMMJOIMMM=ÅçäçåáÉë=ïÉêÉ=ëÅêÉÉåÉÇ=ïáíÜ= íÜÉ=`çcá=Ääçí=Ñçê=ëçäìÄäÉ=éêçíÉáå=ÉñéêÉëëáçåK=cçê=ãçëí=

= QR

í~êÖÉíë=ïÉ=ÅçìäÇ=áÇÉåíáÑó=`çcá=Ääçíë=Åçåí~áåáåÖ=éçëáíáîÉ=ÅçäçåáÉëK=qÜÉ=åìãÄÉê=çÑ=éçëáíáîÉ=ÅçäçåáÉë=ï~ë=îÉêó=í~êÖÉí=ÇÉéÉåÇÉåíI=Ñêçã=îÉêó=ÑÉï=íç=ãçêÉ=íÜ~å=çåÉ=ÜìåÇêÉÇK==

=

=Figure 13 Targets selected for deletion mutagenesis. From the 32 proteins that were used to validate the CoFi blot method, 21 proteins with no or very low levels of

soluble protein expression were selected for deletion mutagenesis. The expression levels were determined by a dot blot of the soluble fraction after a centrifugation.

=cêçã=É~ÅÜ=í~êÖÉí=ïÉ=ëÉäÉÅíÉÇ=OQ=éçëáíáîÉ=ÅäçåÉë=~åÇ=ëìÄàÉÅíÉÇ=íÜÉã=íç=

ÑìêíÜÉê= ÅÜ~ê~ÅíÉêáò~íáçåI= ëã~ää= ëÅ~äÉ= ÉñéêÉëëáçåI= éìêáÑáÅ~íáçå= ~åÇ=ëÉèìÉåÅÉ= ~å~äóëáëK= = qÜÉ= éìêáÑáÉÇ=ã~íÉêá~ä=ï~ë= ~å~äóòÉÇ= Äó= Ççí= Ääçíë=

~åÇ=áå=íÜáë=ëíÉé=ïÉ=ÅçìäÇ=ÇÉíÉÅí=ëçäìÄäÉ=~åÇ=éìêáÑá~ÄäÉ=ã~íÉêá~ä=Ñçê=NQ=çìí=çÑ=íÜÉ=çêáÖáå~ä=ON=í~êÖÉíëK=qÜÉ=ÄÉëí=ÉñéêÉëëáåÖ=ÅçåëíêìÅí=Ñêçã=É~ÅÜ=

í~êÖÉí=ï~ë=~äëç=~å~äóòÉÇ=çå=`ççã~ëëáÉ=ëí~áåÉÇ=papJm^dbK=få=íçí~ä=ïÉ=ÅçìäÇ= ÇÉíÉÅí= ëçäìÄäÉ= ~åÇ= éìêáÑá~ÄäÉ= éêçíÉáå= ÉñéêÉëëáçå= Ñçê= NN= çìí= çÑ=íÜÉ=çêáÖáå~ä=ON=í~êÖÉíëK=qÜÉ=íÜêÉÉ=éêçíÉáåë=ïáíÜ=åç=ÇÉíÉÅí~ÄäÉ=ëçäìÄäÉ=

éêçíÉáå=ÉñéêÉëëáçå=~êÉ=ÉáíÜÉê=Éñíê~ÅÉääìä~ê=çê=ãÉãÄê~åÉ=~ëëçÅá~íÉÇK===tÜÉå= ~å~äóòáåÖ=ïÜÉêÉ= íÜÉ= ëçäìÄäÉ= ÅçåëíêìÅíë= Ü~Ç= íÜÉáê= íê~åëä~íáçå~ä=

ëí~êíI=ïÉ=çÄëÉêîÉÇ=íÜ~í=ã~åó=çÑ=íÜÉ=ëçäìÄäÉ=ÅäçåÉë=ÄÉÖáå=ÅäçëÉ=íç=íÜÉ=éêÉÇáÅíÉÇ=Ççã~áå=ÄçêÇÉêë=EcáÖìêÉ=NRFK=jçëí=äáâÉäóI=íÜÉ=ÅçåëíêìÅíë=íÜ~í=

Ü~îÉ=~=íê~åëä~íáçå~ä=ëí~êí=ïáíÜáå=~=Ççã~áå=ïçìäÇ=ÉñéçëÉ=~ãáåç=~ÅáÇë=íÜ~í= ëÜçìäÇ= ÄÉ= ÄìêáÉÇI= ~åÇ= íÜìë= ïáää= åçí= ÑçäÇ= ÅçêêÉÅíäó= ~åÇ= åçí=éêçÇìÅÉ=~åó=ëçäìÄäÉ=éêçíÉáåK=lå=íÜÉ=çíÜÉê=Ü~åÇI=ÅçåëíêìÅíë=íÜ~í=Ü~îÉ=

~= íê~åëä~íáçå~ä= ëí~êí= ÄÉíïÉÉå= çê= áå= Ñêçåí= çÑ= ~= Ççã~áå=ïçìäÇ= Ü~îÉ= ~=ÜáÖÜÉê=äáâÉäáÜççÇ=çÑ=çÄí~áåáåÖ=ëçäìÄäÉ=éêçíÉáå=ÉñéêÉëëáçåK==

=

= QS

=

==

=Figure 14 Illustration of why deletion mutagenesis may work as an experimental

domain footprinting method. A multi-domain protein is schematically outlined at the top and below is the predicted domain structure obtained from PFAM. There is a

higher likelihood of obtaining soluble constructs (indicated by a triangle) in linker regions. If a deletion mutant starts within a domain, hydrophobic amino acids that

should reside in the core are exposed, increasing the likelihood of misfolding and the formation of inclusion bodies.

=

qÜáë=áë=ëÅÜÉã~íáÅ~ääó=çìíäáåÉÇ=áå=cáÖìêÉ=NQK=tÉ=íÜÉêÉÑçêÉ=ëìÖÖÉëí=íÜ~í=íÜÉ=ê~åÇçã=ÇÉäÉíáçå=~ééêç~ÅÜ=ÅçìäÇ=ëÉêîÉ=~ë=~=íççä=Ñçê=ÉñéÉêáãÉåí~ä=

Ççã~áå= ÑççíéêáåíáåÖK= qÜÉêÉ= ~êÉI= ÜçïÉîÉêI= ÅäçåÉë= íÜ~í= Ü~îÉ= ~=íê~åëä~íáçå~ä= ëí~êí= ïáíÜáå= ~= éêÉÇáÅíÉÇ= Ççã~áå= EcáÖìêÉ= NRFK= qÜÉëÉ=ÅçåëíêìÅíë= ïçìäÇ= åçí= ÄÉ= áÇÉåíáÑáÉÇ= ìëáåÖ= ëí~åÇ~êÇ= ÄáçáåÑçêã~íáÅë=

~ééêç~ÅÜÉëK= fí= ëíáää= êÉã~áåë= íç=ÄÉ= ëÉÉå= áÑ= íÜÉëÉ=éêçíÉáå=ÅçåëíêìÅíë=~êÉ=ìëÉÑìä=Ñçê=ëíêìÅíìê~ä=ëíìÇáÉëK==^åçíÜÉê= áåíÉêÉëíáåÖ= çÄëÉêî~íáçå= áë= íÜ~í= Ñçê= ãçëí= í~êÖÉíë= íÜÉêÉ= ~êÉ=

ÅäçåÉë=íÜ~í=ÉñéêÉëë=ëçäìÄäÉ=éêçíÉáå=~ë=ÑìääJäÉåÖíÜ=çê=ÅäçëÉ=íç=ÑìääJäÉåÖíÜ=éêçíÉáåëK=qÜÉ=Å~ìëÉ=Ñçê=íÜáë=çÄëÉêî~íáçå=áë=åçí=ÇáêÉÅíäó=çÄîáçìë=Äìí=Å~å=

åçí=àìëí=ÄÉ=~ííêáÄìíÉÇ=íç=íÜÉ=ÄáçéÜóëáÅ~ä=ëí~Äáäáíó=çÑ=íÜÉ=í~êÖÉí=éêçíÉáåK=fåëíÉ~Ç= áí= áë= ãçêÉ= äáâÉäó= íÜ~í= íÜÉ= ÉÑÑÉÅí= áë= ÇìÉ= íç= ÅÜ~åÖÉë= áå= íÜÉ=íê~åëÅêáéíáçåI=íê~åëä~íáçå=~åÇ=ÑçäÇáåÖ=ÉîÉåíëK=fí=Ü~ëI=Ñçê=áåëí~åÅÉI=ÄÉÉå=

ëÜçïå=íÜ~í=íÜÉ=êÉÖáçåë=ÇçïåëíêÉ~ã=çÑ=íÜÉ=áåáíá~íáçå=ÅçÇçå=Å~å=~Åí=~ë=íê~åëä~íáçå~ä=ÉåÜ~åÅÉêë=xNORzK==

= QT

===

HP16

HP26

HP27

HP28

HP21

HP24

HP23

HP03

HP08

HP10

HP11

HP14

HP15

HP05

0 50 100 150 200 250 300 350 400 450 500 550 600 650

Residue0 50 100 150 200 250 300 350 400 450 500 550 600 650

Residue

ID Domains as predicted by PFAMDomains as predicted by BIOZONTranslational start of soluble construct

5 2

8

8 4 2

2

5

4

2 2

6

6

2

2

==Figure 15 The domain structure of the 14 proteins in the study was predicted using

PFAM (top) or BIOZON (bottom). The extent of the deletions was determined by

sequencing. The translation starts of purifiable constructs are indicated by triangles or squares.

=

få= ÅçãÄáå~íáçå= ïáíÜ= íÜÉ= çêáÖáå~ä= ëÅêÉÉå= EïÜÉêÉ= NN= í~êÖÉíë= ïÉêÉ=ëçäìÄäÉF=EcáÖìêÉ=NM=~åÇ=cáÖìêÉ=NPF=OO=çìí=çÑ=íÜÉ=PO=Üìã~å=~åÇ=ãçìëÉ=

éêçíÉáåë= ÅçìäÇ= ÄÉ= ÉñéêÉëëÉÇ= áå= bK= ÅçäáK= qÜÉ= ÇÉäÉíáçå= ãìí~ÖÉåÉëáë=ëíê~íÉÖó= íÜìë= ÇçìÄäÉë= íÜÉ= çêáÖáå~ä= ëìÅÅÉëë= ê~íÉK= qÜÉ= ëÉäÉÅíÉÇ= ëçäìÄäÉ=ÅäçåÉë=~äëç=ÅçêêÉëéçåÇ=ïÉääI=Äìí=~êÉ=åçí=áÇÉåíáÅ~äI=íç=éêÉÇáÅíÉÇ=Ççã~áå=

ÄçêÇÉêë= ÑìêíÜÉê= ìåÇÉêäáåáåÖ= íÜÉ= áãéçêí~åÅÉ= çÑ= ÉñéÉêáãÉåí~ä=~ééêç~ÅÜÉëK==

==

= QU

Overproduction of Membrane Proteins ^ë=ãÉåíáçåÉÇI= íÜÉ=ÑìåÅíáçå~ä=~åÇ= áå=é~êíáÅìä~êI= íÜÉ=ëíêìÅíìê~ä=ëíìÇáÉë=çÑ=ãÉãÄê~åÉ= éêçíÉáåë= ä~Ö= ÄÉÜáåÇ= íÜ~í= çÑ= ëçäìÄäÉ= éêçíÉáåëK= qÜáë= Ü~ë=ÄÉÉå=~ííêáÄìíÉÇ= íç= íÜÉ=ìåé~ê~ääÉäÉÇ=ÇáÑÑáÅìäíáÉë= áå=çÄí~áåáåÖ= ëìÑÑáÅáÉåí=

~ãçìåíë= çÑ= ëìáí~ÄäÉ= ë~ãéäÉë= ~åÇ= ïáíÜ= éêçÄäÉãë= ÉåÅçìåíÉêÉÇ= ïÜÉå=íêóáåÖ= íç= Åêóëí~ääáòÉ= ãÉãÄê~åÉ= éêçíÉáåëK= qÜÉ= ÇáÑÑáÅìäíáÉë= áå=

çîÉêéêçÇìÅáåÖ= ãÉãÄê~åÉ= éêçíÉáåë= Ü~îÉ= ÄÉÉå= ~ííêáÄìíÉÇ= íç= ëÉîÉê~ä=ÇáÑÑÉêÉåí=Ñ~ÅíçêëW==íÜÉ=áåëÉêíáçå=ã~ÅÜáåÉêó=Å~å=éçíÉåíá~ääó=ÄÉ=ë~íìê~íÉÇ=xOVzI=íÜÉêÉ=áë=~=äáãáíÉÇ=ëé~ÅÉ=áå=íÜÉ=äáéáÇ=ÄáJä~óÉêI=~åÇ=íÜÉ=éêçíÉáåë=Å~å=

éçíÉåíá~ääó=ÄÉ=íçñáÅ=íç=íÜÉ=ÅÉääë=xNOSzK=bîÉå=áÑ=íÜÉ=éêçíÉáå=áë=éêçÇìÅÉÇ=áå=êÉ~ëçå~ÄäÉ=~ãçìåíë=íÜÉ=åÉñí=ÜìêÇäÉ=íç=çîÉêÅçãÉ=áë=íÜÉ=éìêáÑáÅ~íáçå=

çÑ=íÜÉ=éêçíÉáåK=páåÅÉ=ãÉãÄê~åÉ=éêçíÉáåë=~êÉ=îÉêó=ÜóÇêçéÜçÄáÅ=Äó=íÜÉáê=å~íìêÉI= ÇÉíÉêÖÉåíë=ãìëí= ÄÉ= ìëÉÇ= íç= ëçäìÄáäáëÉ= íÜÉãK= qÜÉêÉ= ~êÉ=ã~åó=

ÇÉíÉêÖÉåíë=~î~áä~ÄäÉI=~ää=ïáíÜ=ÇáÑÑÉêÉåí=éêçéÉêíáÉëI=~åÇ=ìåÑçêíìå~íÉäó=áí=áë=åçí=éçëëáÄäÉ=íç=éêÉÇáÅí=ïÜáÅÜ=ÇÉíÉêÖÉåí=áë=çéíáã~ä=Ñçê=íÜÉ=Éñíê~Åíáçå=

~åÇ= ëí~Äáäáò~íáçå= çÑ= ~= éêçíÉáåK= páãáä~ê= íç= íÜÉ= ï~ó= íÜÉ= ÉñéêÉëëáçå=é~ê~ãÉíÉêë= ~êÉ= ëÅêÉÉåÉÇ= íç= áåÅêÉ~ëÉ= íÜÉ= ÉñéêÉëëáçå= óáÉäÇI= ÇÉíÉêÖÉåíë=

~äëç= Ü~îÉ= íç= ÄÉ= ëÅêÉÉåÉÇ= xNOTzK=tÜÉå= ÉåçìÖÜ= éìêáÑáÉÇ=ã~íÉêá~ä= Ü~ë=ÄÉÉå= çÄí~áåÉÇ= íÜÉ= åÉñí= çÄëí~ÅäÉ= áë= íÜÉ= Åêóëí~ääáò~íáçåK= páåÅÉ= ~= ä~êÖÉ=

Ñê~Åíáçå=çÑ=íÜÉ=éêçíÉáå=áë=ìëì~ääó==ÅçîÉêÉÇ=Äó=ÇÉíÉêÖÉåíëI=çåäó=~=ëã~ää=é~êí=çÑ=íÜÉ=éêçíÉáå=áë=~ÅÅÉëëáÄäÉ=Ñçê=éêçíÉáåJéêçíÉáå=áåíÉê~ÅíáçåëI=ïÜáÅÜ=~êÉ=íÜÉ=ÑçìåÇ~íáçå=Ñçê=çÄí~áåáåÖ=ïÉää=ÇáÑÑê~ÅíáåÖ=Åêóëí~äëK==

=

=Figure 16 Problems encountered during structural studies of integral membrane proteins. (A) Before purification the protein has to be solubilised from the lipid bilayer

using detergents. (B) There is a much lower likelihood of forming well diffracting crystals of membrane proteins since a large fraction of the protein is covered in

detergents.

= QV

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= RN

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ÉñéêÉëëáçå= ~í= ~ää= íç= Ñ~áêäó= ÜáÖÜ= ÉñéêÉëëáçåK= tÜÉå= ëÅêÉÉåáåÖ= íÜÉ=ãìí~ÖÉåÉëáë= äáÄê~êáÉëI=ïÉ= ÅçìäÇ= åçí= ÇÉíÉÅí= ~åó= ëáÖåáÑáÅ~åí= ÉñéêÉëëáçå=Ñçê=Q=çìí=çÑ=íÜÉ=V=í~êÖÉíëI=ëìÖÖÉëíáåÖ=íÜ~í=íÜÉ=ê~åÇçã=ãìí~ÖÉåÉëáë=ÇáÇ=

åçí=áåÅêÉ~ëÉ=íÜÉ=ÉñéêÉëëáçå=äÉîÉä=EcáÖìêÉ=NT=~FK=kçåÉ=çÑ=íÜÉëÉ=Q=í~êÖÉíë=Ü~Ç=~åó=ÉñéêÉëëáçå=~ë=tq=ÅçåëíêìÅíëK=eçïÉîÉêI=`çcá= Ääçíë= çÑ= íÜÉ= êÉã~áåáåÖ= R= äáÄê~êáÉë= ëÜçïÉÇ= ÇÉíÉÅí~ÄäÉ=

ÉñéêÉëëáçå=äÉîÉäëI=ïáíÜ=~=ÜáÖÜ=î~êá~Äáäáíó=ÄÉíïÉÉå=íÜÉ=ëáÖå~ä=ëíêÉåÖíÜ=çÑ=ÇáÑÑÉêÉåí= ÅçäçåáÉëKEcáÖìêÉ= NT= ÄJÇFK= qÜÉ= åìãÄÉê= çÑ= éçëáíáîÉ= ÅçäçåáÉë=

ï~ë= îÉêó= í~êÖÉí= ÇÉéÉåÇ~åíI= Ñêçã= îÉêó= ÑÉï= EcáÖìêÉ= NT= ÄF= íç= ëÉîÉê~ä=ÜìåÇêÉÇK===

= RO

= Figure 17 Examples of CoFi blots of random mutagenesis libraries for four different

membrane proteins. (A) A CoFi blot with no overexpressing clones (B-D) CoFi blots with colonies with different level of overexpression

=qÜÉ=éÉêÑçêã~åÅÉ=çÑ=íÜÉ=ãÉãÄê~åÉ=éêçíÉáå=`çcá=Ääçí=ï~ë=Éî~äì~íÉÇ=Äó=ëÉäÉÅíáåÖ= OQ= ÅäçåÉë= àìÇÖÉÇ= íç= ÉñéêÉëë= ~í= ~= ÜáÖÜ= äÉîÉä= ~åÇ= OQ= ÅäçåÉë=

àìÇÖÉÇ=íç=ÉñéêÉëë=~í=~=äçï=äÉîÉä=Ñêçã=P=ê~åÇçã=ãìí~ÖÉåÉëáë=äáÄê~êáÉëK=qÜÉëÉ= ÅäçåÉë= ïÉêÉ= ÉñéêÉëëÉÇ= áå= ~= äáèìáÇ= ÅìäíìêÉ= ~åÇ= íÜÉ= ~ãçìåí= çÑ=

çîÉêÉñéêÉëëÉÇ= ãÉãÄê~åÉ= éêçíÉáå= ï~ë= ÇÉíÉêãáåÉÇ= Äó= ~= ëã~ää= ëÅ~äÉ=éìêáÑáÅ~íáçå=ÑçääçïÉÇ=Äó=~=Ççí=Ääçí=EcáÖìêÉ=NUFK===

=Figure 18 The performance of the membrane protein CoFi blot. Colonies judged by

the CoFi blot to express at either a high or low level were selected from three different random mutagenesis libraries (MP05, MP06 and MP08). The cells were grown in

liquid culture and the membrane proteins were affinity purified. The level of expression was analyzed by a dot-blot of the purified material.

= RP

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0,5

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5

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MP07 #2 MP07 WT

a b c

d e

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ativ

e in

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MP09 #2 MP09 WTMP09 #1 ==Figure 19 Comparison between the best expressing clones and the corresponding wild type. (a-e) Multiple medium-scale purifications were made for (a) MP03 n=3, (b)

MP04 n=7, (c) MP05 n=4, (d) MP07 n=7 and (e) MP09 n=4. The purified samples were analyzed either by Western blots (a, c and e) or dot blots (b and d) and the

band/dot intensities were measured semi-quantitatively. The wild type intensity was set to 1 and each bar value represents the mean ± s.d. of n experiments.

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