Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli

Bacterial expression systems can greatly facilitate proteinengineering of antibodies. We have developed a system for high-levelexpression of antibodies, antibody fragments, or hybrid antibodieswith novel effector functions in the periplasm of Escherichiacoli. From 5 ml of cells, a simple extraction yields sufficientmaterial for SDS-gel electro-phoresis, detection and characterizationof hapten binding. To demonstrate our system, heavy-chain variableregions and 1 light chains of a mouse anti-NP antibody weresynthesized as hybrid proteins with a bacterial signal peptide(Omp F). Each chain is secreted into the periplasm where processing(cleavage of the signal peptide), folding and heterodimer associationtake place. Periplasmic proteins are released by cold osmoticshock, and hapten-binding activity is easily detected withoutfurther manipulation. The ease of genetic engineering in thissystem will facilitate the production of immunoglobulin derivativesdesigned for specific applications, and expression of thesemolecules in a native state will allow the rapid screening ofcombinatorial libraries and the results of mutagenesis.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.     

Synthesis and expression in Escherichia coli of DNA encoding the murine {lambda}1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen

A 658 bp DNA sequence corresponding to the murine ₁ chain ofa monoclonal antibody, Se 155-4, specific for the Salmonellaserotype B O-antigen, was designed using Escherichia coli preferredcodons and chemically synthesized by ligation of synthetic fragmentsinto a linearized plasmid followed by transformation into E.coli.A synthetic signal peptide (ompA) was fused to express the Lchain as a free polypeptide into the periplasm of E.coli cells.After isolation and purification, heterologous recombinationof the E.coli L chain with mouse H chain gave an active antigen-bindingprotein. The activity was 15–20% when compared to proteincreated by an equivalent association of isolated natural mouseL and H chains as measured by a direct EIA assay. In inhibitionexperiments with the polysaccharide antigen, the two proteinsshowed identical titration curves and 50% inhibition points,indicating comparable K_A values.     

Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli

An advantage of exporting a recombinant protein to the periplasmof Escherichia coli is decreased proteolysis in the periplasmcompared with that in the cytoplasm. However, protein degradationin the periplasm also occurs. It has been widely accepted thatthe thermodynamic stability of a protein is an important factorfor protein degradation in the cytoplasm of E.coli. To investigatethe effect of the thermodynamic stability of an exported proteinon the extent of proteolysis in the periplasm, barnase (an extracellularribonuclease from Bacillus amyloliquefaciens) fused to alkalinephosphatase leader peptide was used as a model protein. A setof singly or doubly mutated barnase variants were constructedfor export to the E.coli periplasm. It was found that the half-lifeof the barnase variants in vivo increased with their thermodynamicstability in vitro. A dominant factor for the final yield ofexported barnase was not exportability but the turnover rateof the barnase variant. The yield of a stabilized mutant wasup to 50% higher than that of the wild type. This suggests thatexporting a protein to the periplasm and using protein engineeringto enhance the stability can be combined as a strategy to optimizethe production of recombinant proteins.     

Engineered turns of a recombinant antibody improve its in vivo folding

Using recombinant antibodies functionally expressed by secretionto the periplasm in Escherichia coli as a model system, we identifiedmutations located in turns of the protein which reduce the formationof aggregates during in vivo folding or which influence cellstability during expression. Unexpectedly, the two effects arebased on different mutations and could be separated, but bothmutations act synergistically in vivo. Neither mutation increasesthe thermodynamic stability in vitro. However, the in vivo foldingmutation correlates with the yield of oxidative folding in vitro,which is limited by the side reaction of aggregation. The invivo folding data also correlate with the rate and activationentropy of thermally induced aggregation. This analysis showsthat it is possible to engineer improved frameworks for semi-syntheticantibody libraries which may be important in maintaining librarydiversity. Moreover, limitations in recombinant protein expressioncan be overcome by single amino acid substitutions.     

Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions

In this report, we describe the expression system that enabledus to produce in Escherichia coli the Fab fragment of a mouseIgM that has previously been shown to inhibit the binding ofIgG to autoantigens by interacting with their variable regions.In our system, both light chain and heavy chain fragments wereput under the control of the malE promoter. The light chainwas fused to the MalE signal sequence, while the heavy chainvariable and first constant region were fused to the alkalinephosphatase signal sequence. In this system, after inductionof the promoter with maltose, the Fab fragment could be detectedin a periplasmic extract of the bacteria by Western blottingand also by ELISA. This Fab fragment was purified on a goatanti-mouse immunoglobulin immunoadsorbent and biotinylated.The Fab fragment produced by E.coli reacted with the trinitrophenyl(TNP) hapten and F(ab')₂ fragments of mouse IgG and these reactivitiescould be specifically inhibited by the corresponding solubleantigens. The dissociation constants of this Fab were 1.65 x10^–6 M for TNP and 5 x 10^–6 M for IgG F(ab')₂ fragments,indicating that the affinity of the Fab fragment compared withthat of the whole IgM molecule was similar for TNP but was lowerfor IgG F(ab')₂ fragments     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptide–heavy chain–linker–lightchain–(His)₅ tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 10–30% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with K_d values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins

The DNA sequence of bovine microsomal cytochrome b₅ has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome b_s in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b₅ cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D ¹HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b₅. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (T_m of 73°C thatis significantly higher than that of ‘tryptic’ ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (T_m 78°C) reflected in the values of H_m, S_mand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome b_s is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein.     

Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1{beta} point mutant modified in a surface loop

Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (T_agg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97—Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the T_m of the global transition. This hypothesisis supported by the location of residue 97 in the long 86–99loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins.     

Selection and characterization of HER2/neu-binding affibody ligands

Affibody® (affibody) ligands that are specific for the extracellulardomain of human epidermal growth factor receptor 2 (HER2/neu)have been selected by phage display technology from a combinatorialprotein library based on the 58 amino acid residue staphylococcalprotein A-derived Z domain. The predominant variants from thephage selection were produced in Escherichia coli, purifiedby affinity chromatography, and characterized by biosensor analyses.Two affibody variants were shown to selectively bind to theextracellular domain of HER2/neu (HER2-ECD), but not to controlproteins. One of the variants, denoted His₆-Z_HER2/neu:4, wasdemonstrated to bind with nanomolar affinity (     

Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli

Rat aldolase C cDNA was inserted in an Escherichia coli expressionvector to construct the rat aldolase C expression plasmid, pRAC42.This plasmid produces active rat aldolase C in the transfectedE.coli host cells. The characteristics of the purified enzyme,e.g. mol. wt, electrophoretic mobilities and kinetic parameters,are indistinguishable from those of authentic rat brain aldolaseC. Three different tetrameric hybrid forms, C₃A, C₂A₂ and CA₃,in addition to C₄ and A₄, were found to be produced in the hostcell when E.coli was co-transfected with expression plasmidsfor rat aldolase C and for human aldolase A. Similarly, thehybrid forms, C₃B, C₂B₂ and CB₃, in addition to C₄ and B₄, werealso produced in the cells when co-transfected with the plasmidsfor rat aldolase C and for human aldolase B.     

Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity

We developed a novel bivalent antibody fragment, the linear(L-) F(ab')₂, comprising tandem repeats of a heavy chain fragmentV_H–C_H1–V_H–C_H1 cosecreted with a light chain.Functional humanized L-F(ab')₂ directed against p185^HER2 wassecreted from Escherichia coli at high titer (100 mg/l) andpurified to homogeneity. The L-F(ab')₂ binds two equivalentsof antigen with an apparent affinity (K_d = 0.46 nM) that iswithin 3-fold of the corresponding thioether-linked F(ab')₂fragment The N-terminal site binds antigen with an affinity(K_d = 1.2 nM) that is 4-fold greater than that for the C-terminalsite, as shown by the comparison of L-F(ab')₂ variants containinga single functional binding site. L-F(ab')₂ has greater antiproliferativeactivity than the thioether-linked F(ab')₂ against the p185^HER2-overexpressingtumor cell line BT474. Linear and thioether-linked F(ab')₂ havevery similar pharmacokinetic properties in normal mice, andtheir serum permanence times are respectively 7- and 8-foldlonger than the corresponding Fab fragment L-F(ab')₂ offersa facile route to bivalent antibody fragments that are potentiallysuitable for clinical applications, and that may have improvedbiological activity compared with thioether-linked F(ab')₂ fragments.     

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli

Bovine somatotropin (bST) was secreted from Escherichia coliat moderate levels of 1–2 µg/ml/OD using expressionvectors in which the bST gene was fused to the lamB secretionsignal. To study the secretion properties of bST in E.coli further,two approaches for modifying the secretion signal were employed.In the first case, fusion proteins were constructed with sixalternative bacterial secretion signals: three from E.coli proteins(HisJ, MalE and OmpA), two from bacteriophage proteins (M13coat protein and PA-2 Lc) and one from the chitinase A proteinof Serratia marcescens. The results, as monitored by Westernblot analysis of both total cell protein and the periplasmicfraction, showed that these changes in the secretion signaldid not significantly affect the secretion properties of bST.In the second approach, a library of random mutations was createdin the lamB secretion signal and 200 independent clones werescreened. The level of secreted bST was determined by growingindividual clones in duplicate in microtiter wells, inducingprotein expression and measuring the bST released by osmoticshock using a particle concentration fluorescent immunoassay.The secretion properties of several novel variants in the LamBsignal peptide are presented.     

Cellulose-binding domains: potential for purification of complex proteins

The endoglucanase CenA and the exoglucanase Cex from Cellulomonasfimi each contain a discrete cellulose-binding domain (CBD),at the amino-terminus or carboxyl-terminus respectively. Thegene fragment encoding the CBD can be fused to the gene of aprotein of interest. Using this approach hybrid proteins canbe engineered which bind reversibly to cellulose and exhibitthe biological activity of the protein partner. Alkaline phosphatase(PhoA) from Escherichia coli, and a ß-glucosidase(Abg) from an Agrobacterium sp. are dimeric proteins. The fusionpolypeptides CenA-PhoA and Abg-CBC_cex are sensitive to proteolysisat the junctions between the fusion partners. Proteolysis resultsin a mixture of homo- and heterodimers; these bind to celluloseif one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoAand CenA-PhoA/PhoA. CBD fusion polypeptides could be used inthis way to purify polypeptides which associate with the fusionpartner.     

Improvement of nutritional value and functional properties of soybean glycinin by protein engineering

Glycinin is one of the predominant storage proteins of soybean.To improve its functional properties (heat-induced gelationand emulsification) and/or nutritional value, the A_1aB_1b proglycininsubunit was modified on the basis of genetically variable domainssuggested from the comparison of amino acid sequences of glycinin-typeglobulins from various legumes and nonlegumes and the relationshipsbetween the structure and the functional properties of glycinin.Thus, nucleotide sequences corresponding to each of the variabledomains were deleted from the cDN A encoding the A_1aB_1b proglycinin,and a synthetic DNA encoding four continuous methionines wasinserted into the cDNA region corresponding to each of the variabledomains. Expression plasmids carrying the modified cDNAs wereconstructed and expressed in Escherichia coli strain JM105.Some of the modified proteins were accumulated as soluble proteinsin the cells at a high level and self-assembled. They exhibitedfunctional properties superior to those of the native glycininfrom soybean, which establishes the possibility of creatingtheoretically designed novel glycinins with high food qualities.     

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment

The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the V_H domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the V_H or the V_L domain, theoptimized version of the affinity peptide termed ‘Strep-tag’allowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidin–alkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.