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.     

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression

Site-directed mutagenesis of the Lactococcus lactis lacR genewas performed to identify residues in the LacR repressor thatare involved in the induction of lacABCDFEGX operon expressionby tagatose-6-phosphate. A putative inducer binding domain locatednear the C-terminus was previously postulated based on homologystudies with the Escherichia coli DeoR family of repressors,which all have a phosphorylated sugar as inducer. Residues withinthis domain and lysine residues that are charge conserved inthe DeoR family were changed into alanine or arginine. The productionof the LacR mutants K72A, K80A, K80R, D210A, K213A and K213Rin the LacR-deficient L.lactis strain NZ3015 resulted in repressedphospho-ß-galactosidase (LacG) activities and decreasedgrowth rates on lactose. Gel mobility shift assays showed thatthe complex between a DNA fragment carrying the lac operatorsand LacR mutants K72A, K80A, K213A and D210A did not dissociatein the presence of tagatose-6-phosphate, in contrast to wildtype LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A,F214R, R216R and R216K) exhibited no gross effects on inducerresponse. The results strongly suggest that the lysines at positions72, 80 and 213 and aspartic acid at position 210 are involvedin the induction of lac operon expression by tagatose-6-phosphate.     

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion

In antigen–antibody interactions, the high avidity ofantibodies depends on the affinity and number of the individualbinding sites. To develop artificial antibodies with multiplevalency, we have fused the single-chain antibody Fv fragmentsto core streptavidin. The resulting fusion protein, termed scFv::strep,was found after expression in Escherichia coli in periplasmicinclusion bodies. After purification of the recombinant productby immobilized metal affinity chromatography, refolding andsize-exclusion FPLC, tetrameric complexes resembling those ofmature streptavidin were formed. The purified tetrameric scFv::strepcomplexes demonstrated both antigen- and biotin-binding activity,were stable over a wide range of pH and did not dissociate athigh temperatures (up to 70°C). Surface plasmon resonancemeasurements in a BIAlite system showed that the pure scFv::streptetramers bound immobilized antigen very tightly and no dissociationwas measurable. The association rate constant for scFv::streptetramers was higher than those for scFv monomers and dimers.This was also reflected in the apparent constants, which wasfound to be 35 times higher for pure scFv::strep tetramers thanmonomeric singlechain antibodies. We could also show that mostof biotin binding sites were accessible and not blocked by biotinylatedE.coli proteins or free biotin from the medium. These sitesshould therefore facilitate the construction of bispecific multivalentantibodies by the addition of biotinylated ligands.     

A method for construction of long randomized open reading frames and polypeptides

A method is presented for construction of randomized open readingframe sequences (ORFs) and gene libraries containing them. Thebuilding blocks for the ORFs were 75 bp long DNA fragments generatedby cloning sequences from a single synthetic oligonucleotidepreparation by bridge mutagenesis. The fragments had the propertythat, regardless of their orientation in the ligated product,the ORF of the construct was maintained. The heterogeneity ofthe ORFs resulted from the random ligation of 2000 differentDNA fragments. The randomized ORFs were cloned downstream fromthe lac promoter in a multicopy plasmid in Escherichia coli.To test the method, a library of 10⁶ clones was constructed.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (K_a 1x10⁹ M^–1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection.     

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.     

Overproduction of bovine {beta}-casein in Escherichia coli and engineering of its main chymosin cleavage site

A cDNA clone containing the entire coding region for bovineß-casein A³ flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192–193 in two ways, namelyfrom Leu–Tyr to Pro–Pro and to Leu–stop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193–209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92– Prol93) was no longerhydrolysed by chymosin at the 192–193 bond.     

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.     

Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned intopKK233-2 vector were recloned from the parent plasmid into thenew procaryotic expression vectors pGFY221N and pBIO52. GeneAF^–B (coding for all amino acids besides phenylalanine)was obtained by ‘cassette mutagenesis’ from geneAB. The plasmid pGFY221N was constructed from pGFY218L by replacingthe PstI by an NcoI site; plasmid pBIO52 was derived from pGFY221Nthrough replacing the 221-bp EoRl/NcoI fragment with a syntheticDNA segment of 52 bp representing the Escherichia coli atpEgene translational initiation region. The genes A, AB, AHB andAF^–B in the vector pGFY221N were expressed with a six-amino-acid-longleader sequence; in pBIO52 the genes were expressed directly.in vitro expression experiments were successful with all thegenes except with the AHB gene integrated into pGFY221N. Inthe E. coli minicell system expression was demonstrated withthe A gene in pGFY221N and the AF^–B and AHB genes in pBIO52.Complete translation of the expressed genes AB, AF^–B andAHB in either the in vitro or in vivo systems could be shownby using ³⁵S-labelled N-terminal methionine and C-terminal cysteine.Both amino acids occur only once in the peptide sequences.     

The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides

The Ecballium elaterium trypsin inhibitor II (EETI-II), a memberof the squash family of protease inhibitors, is composed of28 amino acid residues and is a potent inhibitor of trypsin.Its compact structure is defined by a triple-stranded antiparallelß-sheet, which is held together by three intramoleculardisulfide bonds forming a cystine knot. In order to explorethe potential of the EETI-II peptide to serve as a structuralscaffold for the presentation of randomized oligopeptides, weconstructed two EETI-II derivatives, where the six-residue inhibitorloop was replaced by a 13-residue epitope of Sendai virus L-proteinand by a 17-residue epitope from human bone Gla-protein. EETI-IIand derived variants were produced via fusion to maltose bindingprotein MalE. By secretion of the fusion into the periplasmicspace, fully oxidized and correctly folded EETI-II was obtainedin high yield. EETI-II and derived variants could be presentedon the Escherichia coli outer membrane by fusion to truncatedLpp'–OmpA', which comprises the first nine residues ofmature lipoprotein plus the membrane spanning ß-strandfrom residues 46–66 of OmpA protein. Gene expression wasunder control of the strong and tightly regulated tetA promoter/operator.Cell viability was found to be drastically reduced by high levelexpression of Lpp'–OmpA'–EETI-II fusion protein.To restore cell viability, net accumulation of fusion proteinin the outer membrane was reduced to a tolerable level by introductionof an amber codon at position 9 of the lpp' sequence and utilizingan amber suppressor strain as expression host. Cells expressingEETI-II variants containing an epitope were shown to be surfacelabeled with the respective monoclonal antibody by indirectimmunofluorescence corroborating the cell surface exposure ofthe epitope sequences embedded in the EETI-II cystine knot scaffold.Cells displaying a particular epitope sequence could be enriched10⁷-fold by combining magnetic cell sorting with fluorescence-activatedcell sorting. These results demonstrate that E.coli cell surfacedisplay of conformationally constrained peptides tethered tothe EETI-II cystine knot scaffold has the potential to becomean effective technique for the rapid isolation of small peptidemolecules from combinatorial libraries that bind with high affinityto acceptor molecules.     

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance.     

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     

Elastase substrate specificity tailored through substrate-assisted catalysis and phage display

The catalytic histidine of human neutrophil elastase was replacedwith alanine (H57A) to determine if a substrate histidine couldsubstitute for the missing catalytic group—`substrate-assistedcatalysis'. H57A and wild-type elastase were recovered directlyfrom Pichia pastoris following expression from a synthetic genelacking the elastase pro sequence, thereby obviating the needfor zymogen activation. Potential histidine-containing substratesfor H57A elastase were identified from a phage library of randomizedsequences. One such sequence, REHVVY, was cleaved by H57A elastasewith a catalytic efficiency, k_cat/K_M, of 2800 s^–1 M^–1,that is within 160-fold of wild-type elastase. In contrast,wild-type but not H57A elastase cleaved the related non-histidinecontaining sequence, REAVVY. Ten different histidine-containinglinkers were cleaved by H57A elastase. In addition to the requirementfor a P2 histidine, significant preferences were observed atother subsites including valine or threonine at P1, and methionineor arginine at P4. A designed sequence, MEHVVY, containing thepreferred residues identified at each subsite proved to be amore favorable substrate than any of the phage-derived sequences.Extension of substrate-assisted catalysis to elastase suggeststhat this engineering strategy may be widely applicable to otherserine proteases thereby creating a family of highly specifichistidine-dependant proteases.     

Engineering proteins, subcloning and hyperexpressing oxidoreductase genes

A very efficient system for subcloning and studying proteinsequences, combining previously established elements for hyperexpression,replication and screening, was used to hyperproduce and characterizeseven different products. It expedited the cloning of genes,in a multipurpose recombinant DNA construct, for all the requirementsto study and engineer proteins with a strain of Escherichiacoli. Genes encoding six heme proteins and a flavoprotein havebeen subcloned and expressed to 13–30% of the total cellprotein, greatly facilitating purification and analyses. Threeof the heme proteins and the flavoprotein incorporated prostheticgroups in E.coli, and exhibited the expected activities. Fourof the enzymes have been purified to homogeneity and two ofthese crystallized for X-ray diffraction analysis. A rapid muta-genesisprotocol, based on polymerase chain reactions, was successfullyapplied to clone derivatives of one of these enzymes, cytochromec peroxidase. Thus, this system fulfills all criteria for engineeringproteins in an efficient and concerted manner.     

Molecular mechanics analysis of inhibitor binding to HIV-1 protease

Crystallographic structures of HIV protease with three differentpeptide-mimetic inhibitors were subjected to energy minimizationusing molecular mechanics, the minimized structures analyzedand the inhibitor binding energies calculated. Partial chargeassignment for the hydrogen bonded catalytic aspartk acids,Asp25 and -25', was in good agreement with charge calculationsusing semi-empirical molecular orbital methods. Root mean squaredeviations on minimization were small and similar for both subunitsin the protease dimer. The surface loops, which had the largestB factors, changed most on minimization; the hydrophobic coreand the inhibitor binding site showed little change. The distance-dependentdielectric of D(r) = 4r was found to be preferable to D(r) =r. Distance restraints were applied for the intermolecular hydrogenbonds to maintain the conformation of the inhibitor bindingsite. Using the dielectric of D(r) = 4r, the calculated interactionenergy of the three inhibitors with the protease ranged from–53 to –56 kcal/mol. The groups of the inhibitorswere changed to add or remove a ‘transition state analogue’hydroxyl group, and the loss in energy on the removal of thisgroup was calculated to be 0.9–1.7 kcal/mol. This wouldrepresent 19–36% of the total measured difference in bindingenergy between the inhibitors JG365 and MVT-101.     

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichiacoli has been constructed in one step from long synthetic oligonucleotides.The synthetic gene contains an efficient translation initiationsignal and used codons that are commonly found in E.coli. Uniquerestriction sites are placed throughout the gene. It has beeninserted in a phagemid vector and is expressed from the lacpromoter in E.coli at high efficiency, the soluble heme proteinrepresenting 10% of soluble protein. Two versions of horse heartmyoglobin were produced with aspartic acid or asparagine atresidue 122. Comparison of chromatographic mobilities of thesetwo proteins with authentic horse heart myoglobin identifiedaspartic acid as the correct residue 122. The availability ofthis gene, which is designed to facilitate oligonucleotide mutagenesisor cassette mutagenesis, will allow systematic structure—functionanalysis of horse heart myoglobin.     

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module

Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDS–PAGE ‘snapshot’ analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZ–GST::rFosLZ–GSTheterodimers whereas rJunLZ–GST::rFosLZ and rJunLZ::rFosLZ–GSTformed readily. Furthermore, rJunLZ–GST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (K_a >10⁶ M^-1) with no higher multimeric forms. rFosLZ–GST weaklyassociates beyond a dimer (K_a {small tilde}4x10⁵ M^-1) and rJunLZ–GSTassociates indefinitely (K_a {small tilde}4x10⁶ M^-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired.