A single Fc binding domain-alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity

A recombinant gene fusion was created and cloned using a previouslyconstructed gene encoding a monodomain IgG Fc binding proteinand the gene coding for bacterial alkaline phosphatase. Theconstruct was able to express and secrete a fusion protein thatexhibited both IgG binding and alkaline phosphatase enzymaticactivities. Greater than 60% of the protein demonstrating bothbiological activities was detected from periplasmic space preparations.Nanogram concentrations of the Fc binding-alkaline phosphatasefusion protein allowed primary IgG antibody detection withoutthe use of conjugated secondary antibodies. Removal of the domaincoding for alkaline phosphatase resulted in decreased resistanceof the protein to proteolytic degradation and the loss of IgGFc binding ability. Using affinity-purified fusion protein,the specificity of binding to IgG, IgM and IgA was examined;binding was strong to IgG and barely detectable against IgMor IgA. Affinity for binding of the fusion protein to IgG (k_d= 6.7 x10^-8 M) was determined to be equal to or greater thanpreviously reported for protein A.     

Novel substrate specificity engineered in the arabinose binding protein

The L-arabinose binding protein (ABP) of Escherichia coli naturallybinds L-arabinose and D-galactose with very high affinity and,with reduced affinity, a variety of other sugars that differonly at the C5 position of the pyranose ring.However, thereare stringent specificity requirements at the 1, 2, 3 and 4positions. Based on the high resolution crystallographic structureof the Ugand-protein complex, remodelling of the binding pocketwas attempted to shift the specificity towards Cl-substitutedgalactosides. To create space in the vicinity of the reducingend of bound galactose, four residues, LyslO, Asp90, Thrl47and Leul45, have been mutated for residues with smaller sidechains. Forty-seven mutants containing different combinationsof these mutations were tested by fluorometry for their abilityto bind methylß-D-galactoside (met-ß-Gal)or iso-propyl-ß-D-thio-galactoside (IPTG). Two double-residuemutants carrying Ser at position 147 and Ala or Gly at position90 appeared of particular interest for being able to bind met-ß-Galor IPTG, respectively, and no longer galactose. Fluorescenceexperiments and molecular modelling indicate that the mode ofbinding of the new substrates to the mutant proteins might besimilar to that of the natural ligands to wild-type ABP.     

The prediction and characterization of metal binding sites in proteins

The rational engineering of novel functions into proteins canonly be attempted when the underlying structural scaffold onwhich the new function is displayed and the structure of thetarget protein are both well understood. To introduce functionsmediated by metals it is therefore necessary to identify theprincipal liganding residues for the chosen metal, the requiredarchitecture of the metal-ligand complex and sites within thetarget protein that could accommodate such sites. Here we presenta method that applies structural information from the proteindata bank to the ab initio design and characterization of novelmetal binding sites. The prediction method has been tested on28 metalloprotein structures from the Brookhaven Protein DataBank. It successfully identified >90% of the metal bindingsites. In addition, we have used the method to design and characterizezinc binding sites in two antibody structures. Metal bindingstudies on one of these putative metalloantibodies showed metalbinding, confirming the predictive power of the method.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcalprotein A, designated domain B, was chemically synthesized.This gene was tandemly repeated to give dimeric and tetramericdomain B genes by the use of two restriction enzymes which gaveblunt ends. The genes were highly expressed in Escherichia colito afford a large amount of dimeric and tetrameric domain Bproteins. The single domain B protein was efficiently producedas a fusion protein with a salmon growth hormone fragment. Thefusion protein was converted to monomeric domain B by cyanogenbromide cleavage. The CD spectra of the monomeric, dimeric andtetrameric domain B proteins were essentially the same as thatof native form protein A, showing that their secondary structureswere very similar. The dimeric and tetrameric domain B proteinsformed precipitates with IgG as protein A. This system permitsthe efficient production of mutated single and multiple IgG-bindingdomains which can be used to study structural changes and proteinA–immunoglobulin interactions.     

Analysis of protein conformational characteristics related to thermostability

The thermal stability of proteins was studied, 195 single aminoacid residue replacements reported elsewhere being analysedfor several protein conformational characteristics: type ofresidue replacement; conservative versus nonconservative substitution;replacement being in a homologous stretch of amino acid residues;change in hydrogen bond, van der Waals and secondary structurepropensities; solvent-accessible versus inaccessible replacement;type of secondary structure involved in the substitution; thephysico-chemical characteristics to which the thermostabilityenhancement can be attributed; and the relationship of the replacementsite to the folding intermediates of the protein, when known.From the above analyses, some general rules arise which suggestwhere amino acid substitutions can be made to enhance proteinthermostability: substitutions are conservative according tothe Dayhoff matrix; mainly occur on conserved stretches of residues;preferentially occur on solvent-accessible residues; maintainor enhance the secondary structure propensity upon substitution;contribute to neutralize the dipole moment of the caps of helicesand strands; and tend to increase the number of potential hydrogenbonding or van der Waals contacts or improve hydrophobic packing.     

Characterization of the DNA binding domain of the mouse IRF-2 protein

The DNA binding domain of the interferon regulatory factor-2protein (IRF-2) has been produced and characterized, -chymotrypsindigestion of the purified IRF-2 protein bound to a syntheticbinding site yields a peptide fragment of 14 K in molecularweight. N-terminal analysis of this peptide fragment showedthat its sequence is the same as that of the intact IRF-2. Apeptide fragment of {small tilde} 14 K, IRF-2(113), which correspondsto the N-terminal 113 amino acids of the intact IRF-2 protein,has been expressed in a functional form in Escherichia coli.The first methionine was processed during the expression andthe purified IRF-2(113) thus contains 112 amino acids. DNaseI footprinting and gel retardation assaying showed that IRF-2(113)binds to a synthetic DNA having the consensus binding site andto the upstream regulatory sequence of the IFN-ß geneas intact IRF-2 does. These results showed that this peptidefragment, IRF-2(113), may be a good material for investigationof the DNA binding domain of IRF-2 and of the DNA–proteininteraction.     

Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein

In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837–841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [³H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors.     

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase

A gene coding for the Nereis sarcoplasmic calcium-binding protein(NSCP) was synthesized and expressed in Escherichia coli. Thesequence of the gene was derived from the protein sequence byreverse translation. It possesses a number of unique, regularlyspaced, restriction endonuclease cleavage sites to facilitatefuture site-directed mutagenesis. For the cloning strategy thegene sequence was divided into four parts. Three parts werecloned by ligation of hybridized oligomers and one part by inversePCR. The protein was expressed as a fusion protein with thebacterial chloramphenicol acetyltransferase (CAT), which couldbe easily purified by affinity chromatography. At the junctionof the CAT and NSCP moieties a recognition site for the proteolyticenzyme factor Xa was built in. However, the distance betweenthe moieties appeared to be crucial to warrant cleavage. A kineticanalysis showed that NSCP prepared from the sandworm and theone expressed by E.coli behaved in the same way. This systemprovides a basis for site-specific mutagenesis studies, in orderto elucidate the molecular mechanism of cation binding and concomitantconformational changes     

Destabilizing interactions between the partners of a bifunctional fusion protein

Hybrid MalE–GVP is a bifunctional protein in vitro sinceit binds maltose as protein MalE of Escherichia coli and sinceit is dimeric and specifically binds single-stranded DNA asprotein GVP of phage M13. The oxidation rate of a unique cysteineresidue was used to compare the stabilities of GVP in its freeand hybrid forms, under conditions where MalE was either foldedor unfolded by a denaturing agent. The results showed that boththe covalent link and tertiary non-covalent interactions betweenMalE and GVP destabilized GVP in MalE–GVP. To test whetherGVP had identical structures in its free and hybrid forms, mutationswere used as local conformational probes. The effects of thesemutations on the capabilities of MalE–GVP to dimerizeand to bind single-stranded DNA were assayed in vitro. Theywere compatible with the effects of the same mutations on theglobal activity of free GVP in vivo and with the effects thatcould be predicted from the known data on free GVP, in particularits crystal structure. Thus, one partner of a hybrid proteincan be destabilized by the other partner while maintaining itsstructural and functional characteristics.     

Grafting of discontinuous sites: a protein modeling strategy

A strategy for modeling continuous as well as discontinuoussites in protein structures has been developed. Central to thismodeling strategy is the search algorithm of FITSITE, a programto search a given target structure for suitable combinationsof backbone positions mirroring as closely as possible the geometricrelationships of a source structural motif of interest. Alltarget sites detected by FITSITE are further refined to mimicthe source geometry. The side-chain rotamer library conceptfails to precisely describe side chains involved in coordinativebonding (e.g. metal binding sites). Therefore an algorithm usingdetailed database bonding parameter information was appliedfor the side-chain construction. The FITSITE program and thesubsequent processing of the program output are presented ina test case. The Rop protein, a four-helix bundle structure,served as the target protein. It was searched for candidatesites to model a variety of metal binding sites, with structuresextracted from Brookhaven Protein Database entries. The preliminaryprotein models were investigated for structural overlaps withneighboring residues by interactive computer graphics; if required,additional changes were performed. A set of parameters for energyminimization with AMBER (including metal ions) was developed,and the completed Rop variants were energy minimized. Finally,12 potentially metal binding Rop variants were selected forproduction via genetic engineering.     

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of Protein A from Staphylococcus aureus

A fusion protein, consisting of the N-terminal 81 amino acidsfrom an inactive bovine DNase I (Q38,E39–E38,Q39) andtwo sequential synthetic IgG-binding domains based upon domainB of Protein A from Staphylococcus aureus has been shown tobind to porcine IgG with a similar affinity and pH profile toProtein A. The same residue in each B domain (Tyr111 and Tyr169)has been mutated by cassette mutagenesis to Ser, Glu, His, Lysor Arg and the effect of the mutation on binding interactionswith porcine IgG investigated. The evidence presented suggeststhat the interactions at the B domain are highly sensitive tothe presence of a charged residue.     

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK,has been constructed which greatly facilitates the insertionand removal of mutant enzyme genes by cleavage at newly introducedBamtHI sites. This vector has been used to prepare mutant proteinin appreciable (100 mg) quantities for use in kinetic, crystaUographicand NMR experiments. Aspartate 372 is an invariant amino acidresidue in genes known to code for a functionally active PGK.The function of this acidic residue appears to be to help desolvatethe magnesium ion compfexed with either ADP or ATP when thissubstrate binds to the enzyme. Both crystallographk and nuclearmagnetic resonance experiments show that the replacement ofthe residue with asparagine has only minimal effects on theoverall structure. The substitution of the charged carboxylgroup with that of the neutral amide affects the binding ofthe nucleotide substrate as predicted but not, as might havebeen expected, the binding of 3-phospho-glycerate. The overallvelocity of the enzymic reaction (V_max) is reduced 10-fold bythe substitution of aspartic acid 372 by an asparagine residue(D372N). This reduction in V_max is considerably less than onewould expect from its known position within the structure ofthe enzyme. This result therefore poses questions about ourunderstanding of charged groups at the active centres of enzymesand of the reason for their apparent conservation.     

Production and properties of a factor X-cellulose-binding domain fusion protein

A fusion protein, FX–CBD_Cex, which comprises factor Xwith a cellulose-binding domain (CBD_Cex) fused to its C-terminus,was produced in BHK cells. It was purified from the culturemedium by affinity chromatography on cellulose. FX–CBD_Cexcould be activated to FXa–CBD_Cex with Russell viper venom.FXa–CBD_Cex was as active as FXa against a chromogenicsubstrate and against proteins containing the Ile–Glu–Gly–Argsequence hydrolysed by FXa. FXa–CBD_Cex retained its activitywhen adsorbed to cellulose.     

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

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.     

Redesigning a sweet protein: increased stability and renaturability

Monellin is one of two natural proteins from African berrieswith potent sweet taste. Monellin is the smaller of the two,and consists of two peptides. The protein loses sweetness whenheated above 50°C under acidic pH. Based on the crystalstructure of monellin we have fused the two chains into a singlechain using several different linkers copied and ‘transplanted’from the same molecule. One of the newly designed proteins isas potently sweet as the natural one, is more stable upon temperatureor pH changes, and renatures easily even after heating to 100°Cat low pH.     

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.