The effects of induction conditions on production of a soluble antitumor sFv in Escherichia coli

CC49 is a second generation monoclonal antibody(mAb) with highaffinity to a pancarcinoma antigen, TAG-72. A single-chain Fv(sFv)ofCC49 may have a role in managing human carcinomas. Most reportedsFvs have been expressed as insoluble products that must besolubilized and renatured. Soluble sFv expression is advantageousas activity can be assayed directly from the periplasmic fraction.Also, gene-level immunoconjugates may not be amenable to refoldingprotocols. Using a vector that carries the tac promoter andomp A signal, we have examined the effects of four variableson the expression and accumulation of soluble CC49 sFv: (i)linker sequence joining VL and VH, (ii) isopropylthio-ß-D-galactosideconcentration for induction, (iii) temprature, and (iv) theaddition of nonmetabolizable sugars to the medium. We have beenable to demonstrate, using rapidly prepared periplasmic extracts,that the yield of soluble sFv improves by the addition of 0.4Msucrose to the medium and by inducing expression with a verylow concentration of IPTG (0.02–0.03 mM). Under theseinduction conditions periplasmic extracts demonstrate increasedexpression of the sFv, as shown by the larger amount of a 27kDa band on SDS-polyacrylamide gel, and an increased abilityto inhibit binding of the mAb CC49 to immobilized tumor extracts.     

Linker modification introduces useful molecular instability in a single chain antibody

SCA 4-4-20/212, is a recombinant single chain antibody directedagainst fluorescein (Fl) composed of the variable light (V_L)and variable heavy (V_H) domains of the monoclonal antibody 4-4-20,tethered by a 14 amino acid linker. Binding of SCA 4-4-20/212to Fl quenches its fluorescence, thus enabling the distinctionbetween bound and free Fl. This was used to follow antibodydenaturation which followed a two-step process: rapid selectedand restricted denaturation followed by slow and progressivedenaturation. This two-phase phenomenon might reflect selectivesusceptibility of the CDR loops to denaturation. Furthermore,a new SCA, SCA 4-4-20/9, was constructed by site-directed mutagenesisof SCA 4-4-20/212 using PCR methodology. SCA 4-4-20/9 was similarto SCA 4-4-20/212, but for a nine residue linker. The two SCAswere compared for Fl binding, heat stability, the effect ofdenaturing agents and susceptibility to proteolysis. The modificationof the linker caused a general conformational rearrangementin the SCA molecule, rendering it more sensitive to denaturationand proteolysis. This molecular instability may find utilityin the application of SCAs in analytical systems or as the recognitioncomponent in biosensors.     

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli

Single-chain antibodies consist of the variable, antigen-bindingdomains of antibodies joined to a continuous polypeptide bygenetically engineered peptide linkers. We have used the flexibleinterdomain linker region of a fungal cellulase to link togetherthe variable domains of an anti-2-phenyloxazolone IgGl and showhere that the resulting single-chain antibody is efficientlysecreted and released to the culture medium of Escherichia coli.The yield of affinity-purified single-chain antibody is 1 -2mg/1 of culture medium and its affinity and stability are comparableto those of the corresponding native IgG.     

Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.     

Engineering of DNA binding proteins into site-specific cutters: reactivity of Trp repressor-1,10-phenanthroline chimeras

Trp repressor (TrpR) can be converted into a site-specific nucleateby chemical modification of the cysteine mutants TrpR D46C orTrpR E49C with 5-iodoacetamido-l,10-phenanthroline (OP). Inthe presence of cupric ion and 3-mercaptopropionic acid, TrpR-regulatedoperators are cleaved. The properties of these semisyntheticscission reagents have been compared. The E49C construct cleavesefficiently at two sites within the operator and the D46C cleavesat multiple sites. Molecular modeling indicates that the reasonfor the focused reactivity of E49C is that the OP is rigidlyoriented in the protein-DNA complexes whereas the OP can adoptseveral orientations in TrpR D46C. Mutations and reaction conditionsthat increase the affinity of the repressor enhance the scissionefficiency which approaches 100% within the acrylamide matrix.TrpR E49C-OP smoothly cleaves the trpEDCBA operator in a plasmidin a reaction dependent on the corepressor L-tryptophan. Inthe absence of tryptophan, non-specific cleavage of the plasmidis observed under the same conditions. Therefore, tryptophannot only directs cleavage to a specific site but also blocksit at non-specific sites. The analysis of the cleavage patternof the trpEDCBA operator provides strong evidence for the tandembinding model in which protein-protein interactions stabilizebinding on the DNA. TrpR E49C-OP should serve as the basis forthe engineering of a family of highly specific semisyntheticscission reagents.     

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.     

Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv')have been engineered using model sFv proteins based upon the26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonalantibodies. As part of the 741F8 sFv construction process, thePCR-amplified 741F8 V_H gene was modified in an effort to correctpossible primer-induced errors. Genetic replacement of the N-terminalp-strandsequence of 741F8 VH with that from the FR1 of anti-c-erbB-2520C9 VH resulted in a dramatic improvement of sFv folding yields.Folding in urea-glutathione redox buffers produced active sFv'with a protected C-terminal sulfhydryl, presumably as the mixeddisulfide with glutathione. Disulfide-bonded (sFv')₂ homodimerswere made by disulfide interchange or oxidation after reductiveelimination of the blocking group. Both 26–10 (sFv')₂and 741F8 (sFv')₂ existed as stable dimers that were well behavedin solution, whereas 741F8 sFv and sFv' exhibited considerableself-association. The 741F8 sFv binds to the extracellular domain(ECD) of the c-erbB-2 oncogene protein, which is often overexpressedin breast cancer and other adenocarcinomas. The recombinantECD was prepared to facilitate the analysis of 741F8 bindingsite properties; the cloned ECD gene, modified to encode a C-terminalSer-Gly-His₆ peptide, was transfected into Chinese hamster ovarycells using a vector that also expressed dihydrofolate reductaseto facilitate methotrexate amplification. Optimized cell linesexpressed ECD-His₆ at high levels in a cell bioreactor; afterisolation by immobilized metal affinity chromatography, finalECD yields were as high as 47 mg/l. An animal tumor model complementedphysicochemical studies of 741F8 species and indicated increasedtumor localization of the targeted 741F8 (sFv')₂ over othermonovalent 741F8 species.     

Zinc ions bound to chimeric His4/lactate dehydrogenase facilitate decarboxylation of oxaloacetate

A chemically synthesized DNA linker coding for a peptide fragmentthat contains four histidines was fused in-frame to the 5'-endof the Bacillus stearothermophilus lactate dehydrogenase gene.The gene product, His₄/lactate dehydrogenase, could be purifiedto homogeneity using either immobilized metal (Zn²⁺)-affinitychromatography or affinity chromatography on oxamate agarose.The stability against heat and urea for the modified enzymeswas decreased as compared to the native lactate dehydrogenasebut could be increased if zinc ions were present during thedenaturation. In the presence of zinc ions the His₄/lactatedehydrogenase could catalyse the sequential reaction from oxaloacetateto L-lactate, hence operating as a semi-synthetic bifunctionalenzyme. A small increase in the apparent secondorder rate constant(k_cat/K_m) of the coupled reaction was observed as compared toa corresponding system with native lactate dehydrogenase.     

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolatedehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolatesynthetase was engineered to contain a prokaryotic ribosomebinding site and was expressed under the bacteriophage T7 RNApolymerase promoter in Escherichia coli. Site-directed mutagenesiswas used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase(D/C) domain as amino acid residues 1–301, and the synthetase(Syn) domain as residues 304–935. Both domains formedactive enzymes thereby demonstrating their ability to fold independently.The full-length enzyme, D/C and Syn domains were expressed atlevels 4-, 55- and 3-fold higher than the specific activitiesfound in liver. Additional mutagenesis and independent expressionof domains further defined the interdomain region to includeamino acids 292–310. The D/C domain was purified to homogeneityby a single affinity chromatographic step, and the full-lengthprotein in a twostep procedure. The kinetic properties of theD/C domain appear unaltered from those of the trifunctionalenzyme.     

Analysis of the structure of Pseudomonas glumae lipase

The lipase produced by Pseudomonas glumae is monomeric in thecrystalline state and has a serine protease-like catalytic triad;Ser87-His285-Asp263. The largest domain of the protein resemblesclosely a subset of the frequently observed /ß-hydrolasefold and contains a well-defined calcium site. This paper describesstructural analysis of this protein, focusing on (i) structuralcomparison with the lipase from Geotrichum candidum, (ii) theprobable nature of the conformational change involved in substratebinding and (iii) structural variations amongst the family ofPseudomonas Upases. This analysis reveals similarities betweenP.glumae lipase and G.candidum lipase involving secondary structuralelements of the hydrolase core and the loops carrying the catalyticserine and histidine residues. A possible functional equivalencehas also been identified between parts of the two moleculesthought to be involved in a confonmational change. In addition,determination of the structure of P.glumae lipase has allowedrationalization of previously reported protein engineering experiments,which succeeded in improving the stability of the enzyme withrespect to proteolysis.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

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.     

FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolylisomerase that binds the macrolides FK506 and rapamycin. Wehave examined the role of the binding pocket residues of FKBP12in protein–ligand interactions by making conservativesubstitutions of 12 of these residues by site-directed mutagenesis.For each mutant FKBP12, we measured the affinity for FK506 andrapamycin and the catalytic efficiency in the cis–transpeptidyl-prolyl isomerase reaction. The mutation of Trp59 orPhe99 generates an FKBP12 with a significantly lower affinityfor FK506 than wild-type protein. Tyr26 and Tyr82 mutants areenzymatically active, demonstrating that hydrogen bonding bythese residues is not required for catalysis of the cis–transpeptidyl-prolyl isomerase reaction, although these mutationsalter the substrate specificity of the enzyme. We conclude thathydrophobic interactions in the active site dominate in thestabilization of FKBP12 binding to macrolide ligands and tothe twisted-amide peptidyl-prolyl substrate intermediate.     

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

Site-directed mutants of the cAMP receptor protein-DNA binding of five mutant proteins

Oligonucleotide-directed mutagenesis was employed to generatemutants of the cAMP receptor protein (CRP) of Escherichia coli.The mutant proteins were purified to homogeneity and testedfor stability and DNA binding. It is shown that mutations atthe position of Arg¹⁸⁰ abolish specific DNA binding, whereasthose at the position Arg¹⁸⁵ have very little effect. Both positionshave previously been implicated as crucial for the specificinteraction between CRP and DNA. The Ser¹²⁸ Ala mutant showsa slight reduction in DNA binding affinity relative to wild-type.All mutants investigated show similar stability profiles towild-type CRP with respect to thermolysin proteolysis as a functionof temperature.     

Structural similarities in glucoamylases by hydrophobic cluster analysis

The model of the catalytic domain of Aspergillus awamori var.X100 glucoamylase was related to 14 other glucoamylase proteinsequences belonging to five subfamilies. Structural featuresof the different sequences were revealed by multisequence alignmentfollowing hydrophobic cluster analysis. The alignment agreedwith the hydrophobic microdomains, normally conserved throughoutevolution, evaluated from the 3-D model. Saccharomyces and Clostridiumglucoamylases lack the -helix exterior to the catalytic domain.A different catalytic base was found in the Saccharomyces glucoamylasesubfamily. The starch binding domain of fungal glucoamylaseshas identical structural features and substrate interactingresidues as the C-terminal domain of models of Bacillus circulanscyclodextrin glucosyltransferases. Three putative N-glycosylationsites were found in the same turns in glucoamylases of differentsubfamilies. O-Glycosylation is present at different levelsin the catalytic domain and in the linker between the catalyticand starch binding domains.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Taq DNA polymerasespecific binding protein (affibody) was investigated by an -helixshuffling strategy. The primary affibody was from a naive combinatoriallibrary of the three-helix bundle Z domain derived from staphylococcalprotein A. A hierarchical library was constructed through selectivere-randomization of six amino acid positions in one of the two-helices of the domain, making up the Taq DNA polymerase bindingsurface. After selections using monovalent phage display technology,second generation variants were identified having affinities(K_D) for Taq DNA polymerase in the range of 30–50 nM asdetermined by biosensor technology. Analysis of binding dataindicated that the increases in affinity were predominantlydue to decreased dissociation rate kinetics. Interestingly,the affinities observed for the second generation Taq DNA polymerasespecific affibodies are of similar strength as the affinitybetween the original protein A domain and the Fc domain of humanimmunoglobulin G. Further, the possibilities of increasing theapparent affinity through multimerization of affibodies wasdemonstrated for a dimeric version of one of the second generationaffibodies, constructed by head-to-tail gene fusion. As comparedwith its monomeric counterpart, the binding to sensor chip immobilizedTaq DNA polymerase was characterized by a threefold higher apparentaffinity, due to slower off-rate kinetics. The results showthat the binding specificity of the protein A domain can bere-directed to an entirely different target, without loss ofbinding strength.     

Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1

The evolutionally conserved aspartyl residues (Asp57, Asp98and Asp152) in human glutathione S-transferase P1-1 were replacedwith alanine by site-directed mutagenesis to obtain the mutants(D57A, D98A and D152A). The replacement of Asp98 with alanineresulted in a decrease of the affinity for S-hexyl-GSH-agarose,a 5.5-fold increase of the K_m^GHS and a 2.9-fold increase ofthe I₅₀ of S-hexyl-GSH for GSH–CDNB conjugation. Asp98seems to participate in the binding of GSH through hydrogenbonding with the -carboxylate of the -glutamyl residue of GSH.The k_cat of D98A was 2.6-fold smaller than that of the wild-type,and the pK_a of the thiol group of GSH bound in D98A was {smalltilde}0.8 pK units higher than those in the wild-type. Asp98also seems to contribute to the activation of GSH to some extent.On the other hand, most of the kinetic parameters of D57A andD152A were similar to those of the wild-type. However, the thermostabilitiesof D57A and D152A were significantly lower than that of thewild-type. Asp57 and Asp152 seem to be important for maintainingthe proper conformation of the enzyme.     

Ligand requirements for Ca2+ binding to EGF-like domains

Site-specific mutagenesis studies of the first epidermal growthfactor-like (EGF-like) domain of human clotting factor IX suggestthat the calcium-binding site present in this domain (dissociationconstant K_d=1.8 mM at pH 7.5 and ionic strength I=0.15) involvedthe carboxylate residues Asp47, Asp49 and Asp64. To furthercharacterize the ligands required for calcium binding to EGF-likedomains, two new mutations, Asp47 - Asn and Asp49 - Asn, wereintroduced into the domain by peptide synthesis. ¹H-NMR spectroscopywas used to obtain the dissociation constants for calcium bindingto these mutations. Calcium binding to the Asp49- Asn modifieddomain is only mildly affected (K_d=6 mM, I=0.15), whereas bindingto the Asp47- Asn modified domain is severely reduced (K_d=42mM, I=0.15). From these data, it is proposed that the anionicoxygen atoms of the side chains of residues 47 and 64 are essentialfor calcium binding, whereas the side chain ligand for calciumat residue 49 can be a carboxyamide oxygen. As a control, theintroduction of the modification Glu78- Asp in a region of thedomain not believed to be involved in calcium binding had verylittle effect on the K_d for calcium (K_d=2.6 mM, I=0.15). Finally,the effect of an Asp47- Gly substitution found in the naturalhaemophilia B mutant, factor IX_Alabama, was investigated. Thispeptide has a markedly reduced affinity for calcium (K_d=37 mM,I=0.15), suggesting that the defect in factor IX_Alabama is dueto impaired calcium binding to its first EGF-like domain.     

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.