Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus

A novel protein able to bind with high affinity to the Fc fragmentof IgG from a variety of animals has been produced by a genesynthesis approach. The IgG binding is accomplished by the presenceof a single or two consecutive domains based upon domain B fromprotein A of Staphylo-coccus aureus. The IgG-binding moietyis fused to a peptide containing 21, 53 or 81 amino acids derivedfrom the N-terminus of bovine DNase I. The latter is presentto guide the expression of the protein in Escherichia coli intoan inclusion body. This facilitates the high expression andrecovery of the IgG-binding domains. The binding activity ofthis fusion protein is very close to that of the native proteinA. Site-directed mutagenesis of the fusion protein and subsequentidentification of changed binding interactions is reported.     

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.     

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps

The gene coding for the tyrosine protein kinase domain of v-fpswas subcloned into a plasmid vector expressing glutathione-S-transferase(GST). This new vector expresses a fusion protein in Escherichiacoli composed of the kinase domain linked with GST at the N-terminus(GST-kin). A portion of the total expressed protein was solubleupon cell lysis and was purified by affinity chromatographyusing glutathione cross-linked agarose. GST-kin (M_r 57 000)is a phosphoprotein as judged by ³²P autoradiography, consistentwith the known autophosphorylation site within the kinase core[Weinmaster et aL (1984) Cell, 37, 559–568]. Cleavageof the fusion protein with thrombin and purification on phosphocelluloseresin yielded the pure kinase domain (M_r 33 000). The activityof the kinase domain is indistinguishable from that of GST-kinusing the peptide substrate EEEIYEEIE, indicating that Nterminalfusion has no effect on the kinase domain. GSTkin phosphorylatesa second peptide, EAEIYEAIE, with improved catalytic efficiency.Initial velocity data are consistent with a random bireactantmechanism with no substrate synergism observed in the ternarycomplex. Steady-state kinetic analyses reveal that this peptideis phosphorylated, with a k_cat of 3.6 s^–1, a K_peptideof 500 µM and a K_ATP of 250 µM. The expression,purification and preliminary kinetic analysis of the kinasedomain of v-fps provide the first step in the application ofstructurefunction studies for this oncoprotein     

Overproduction, preparation of monoclonal antibodies and purification of E.coli asparagine synthetase A

In order to explore the structure-function relationship of theEscherichia coli asparagine synthetase A it was necessary todevise a system for overexpression of the gene and purificationof the gene product. The E.coli asparagine synthetase A structuralgene was fused to the 3' end of the human carbonic anhydraseII structural gene and overexpressed in E.coli. The gene product,a 66 kDa fusion protein, which exhibited asparagine synthetaseactivity, was purified in a single step by affinity chromatographyand used as the antigen for the production of monoclonal antibodies.The monoclonal antibodies were screened by ELISA. Colonies werechosen which were positive for purified fusion protein and negativefor purified human carbonic anhydrase II. The E.coli asparaginesynthetase A gene was then overexpressed and the gene productwas used without purification for the final screen. The antibodiesselected were used for immunoaffinity chromatography to purifythe recombinant overexpressed E.coli asparagine synthetase A.Thus, a procedure is now available so that asparagine synthetaseA can be purified to homogeneity in a single step.     

Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the productof the Escherichia coli aroA gene, has been overproduced inE.coli BL21(DE3) under the control of the T7 gene 10 promoterand ribosome binding site, to a level of {small tilde}50% oftotal cell protein. EPSP synthase is the primary target of thepost-emergence herbicide, glyphosate, commonly known as Roundup^TM.A simple two step purification is described, which results in99% pure homogeneous protein (as determined by PAGE). The integrityof the protein has been compared with previously characterizedprotein from .E.coli AB2829(pKD501) by determination of itskinetic parameters, N-terminal protein and DNA sequences, aminoacid analysis and ¹³C-NMR spectroscopy. This new overproducingstrain readily provides the gram quantities of highly pure proteinrequired for NMR studies of the active site and the developmentof novel time-resolved solid-state NMR techniques currentlyunderway in this laboratory.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system

In an attempt to introduce a large peptide that is not normallytranslocated across membranes into the cytosol of eukaryoticcells, we created a new chimeric protein termed CEDH betweenPseudomonas aeruginosa exotoxin A (ETA) and a variant enzymeof Mus musculus dihydrofolate reductase (DHFR) with reducedaffinity for antifolates, ETA^1–413.DHFR^1–187.ETA^609–613.We have defined, genetically constructed and expressed the chimericprotein in Escherichia coli. We showed that the CEDH chimericprotein, purified to homogeneity on an immunoaffinity resin,confers a methotrexate-resistant phenotype to Chinese hamsterovary cells. Furthermore, the chimeric protein allowed the growthof dihydrofolate reductase-deficient Chinese hamster ovary cellsin the absence of hypoxanthine and thymidine. These resultsdemonstrated that the chimeric protein exhibited enzyme activityand possessed the tightly folded native structure, and thatthe DHFR protein can be selectively internalized and translocatedvia domains of exotoxin A. These data show that the ETA systemis an efficient system for the delivery of a variety of largepolypeptides into the cytosol without stress to the target cells,and extends the use of this delivery system to proteins thatare not normally translocated across membranes.     

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.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu²⁷) analogueof human growth hormone releasing hormone-Gly⁴⁵ [(Leu²⁷GHRH-Gly⁴⁵]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 15–20% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu²⁷)hGHRH-Gly⁴⁵was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference.     

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.     

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.     

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.     

A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-{beta}-galactosidase

The lacG gene encoding the 6-phospho-ß-galactosidase(E.C.3.2.1.85) of Staphylococcus aureus was fused to the proteinA gene in the plasmid pRIT2T. Escherichia coli cells containingthis plasmid produce a fusion protein with both IgG bindingand 6-phospho-ß-galactosidase activities after heatinduction. The recombinant gene was overexpressed and the hybridprotein was purified to homogeneity in high yield. The chimericprotein was shown to have almost identical enzymatic characteristicsto pure 6-phospho-ß-galactosidase. This result leadsto the conclusion that a free N-terminus of the 6-phospho-ß-galactosidaseis not required for biological activity. The hybrid proteinof protein A and 6-phospho-ß-galactosidase was usedas an enzyme conjugate in enzyme-linked immunosorbent assays(ELISA). The experiments presented demonstrate that the 6-phospho-ß-galactosidaseis a suitable fusion partner in various diagnostic applicationswhere an unique biological activity is required.     

Enzyme IIIlac of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis of histidine residues, biochemical characterization and 1H-NMR studies

The lactose-specific pbosphocarrier protein enzyme III of thebacterial phosphoenol-pyruvate-dependent phosphotransferasesystem of Staphylococcus aureus was modified by sitespecificmutagenesis on the corresponding lacF gene in order to replacethe histidine residues 78 and 82 of the amino acid sequencewith a serine residue. Wild-type and both mutant genes wereoverexpressed in Escherichia coli and the gene products werepurified to homogeneity. The conformation of wild-type and mutantproteins were monitored by ¹H-NMR spectroscopy. In vitro phosphorylationstudies on mutant lactose-specific enzyme III, as well as evidencefrom NMR spectroscopy, lead to the conclusion that His78 isthe activesite for phosphorylation of lactose-specific enzymeIII by phospho-HPr (histidine-containing protein). The roleof His82 probably is the enhancement of velocity and efficiencyof the phosphotransfer from lactose-specific enzyme in to lactosespecifkenzyme II. This result refutes the conclusion of former workbased on data by protelytk cleavage and sequencing of the ³²P-labeledpeptide of lactose-specific enzyme DTI that His82 is the active-sitefor phosphorylation.     

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling

Starting from a synthetic modular gene (infA^*) encoding Escherichiacoli translation initiation factor IF1, we have constructedmutants in which amino acids are deleted from the carboxyl terminusor in which His29 or His34 are replaced by Tyr or Asp residues.The mutant proteins were overproduced, purified and tested invitro for their properties in several partial reactions of thetranslation initiation pathway and for their capacity to stimulateMS2 RNA-dependent protein synthesis. The results allow for theconclusion that: (i) Arg69 is part of the 30S ribosomal subunitbinding site of IF1 and its deletion results in the substantialloss of all IF1 functions; (ii) neither one of its two histidinesis essential for the binding of IF1 to the 30S ribosomal subunit,for the stimulation of fMet-tRNA binding to 30S or 70S ribosomalparticles or for MS2 RNA-dependent protein synthesis; but (iii)His29 is involved in the 50S subunit-induced ejection of IF1from the 30S ribosomal subunit.     

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.     

An internal cellulose-binding domain meidates adsorption of an engineered bifunctional xylanase/cellulase

A chimeric xylanase/endoglucanase (XynCenA) with an internalcellulose-binding domain was constructed by fusing the Bacillussubtilis xyn gene fragment to the 5'-end of the Cellulomonasfimi cenA. A polyhistidine-encoding sequence was also fusedto the 5'-end of the xyn gene. The gene fusion was overexpressedin Escherichia coli and the fusion poly-peptide purified fromthe cell extracts using the polyhistldine tail. The hybrid proteinbehaved like the parental endo-glucanase or xylanase when assayedon a number of soluble and insoluble cellulosic substrates orxylans. The presence of two distinct active sites and the internalcellulose-binding domain did not significantly affect the hydrolysisof any of these substrates. However, the fusion protein exhibiteda strong affinity for both mkrocrystaUine cellulose (Avicel)and regenerated chitin. Like the parental endoglucanase, boundXynCenA could not be duted from these porysaccharides with eitherlow or high salt buffer or distilled water. More stringent conditions,such as 1% SDS or 8 M guanidinium hydro-chloride, fully desorbedthe protein. The fusion protein did not adsorb significantlyto insoluble xylan     

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

The A+T-rich genome of the human malaria parasite Plasmodiumfalciparum encodes genes of biological importance that cannotbe expressed efficiently in heterologous eukaryotic systems,owing to an extremely biased codon usage and the presence ofnumerous cryptic polyadenylation sites. In this work we haveoptimized an assembly polymerase chain reaction (PCR) methodfor the fast and extremely accurate synthesis of a 2.1 kb Plasmodiumfalciparum gene (pfsub-1) encoding a subtilisin-like protease.A total of 104 oligonucleotides, designed with the aid of dedicatedcomputer software, were assembled in a single-step PCR. Theassembly was then further amplified by PCR to produce a syntheticgene which has been cloned and successfully expressed in bothPichia pastoris and recombinant baculovirus-infected High Five^TMcells. We believe this strategy to be of special interest asit is simple, accessible and has no limitation with respectto the size of the gene to be synthesized. Used as a systematicapproach for the malarial genome or any other A + T-rich organism,the method allows the rapid synthesis of a nucleotide sequenceoptimized for expression in the system of choice and productionof sufficiently large amounts of biological material for completemolecular and structural characterization.     

Conversion of the guanine nucleotide binding sites of ras protein resulting in the reduction of base specificity

A gene coding for the novel ras protein, p21^x, in which thedomains of guanine binding and phosphate binding were exchanged,was constructed and expressed in Escherichia coli. The geneproduct, p21^x, showed GTP binding activity, but no GPTase activity.In addition, p21^x revealed binding activity toward ATP and CTP.In a competitive binding assay, [³H]GTP binding to p21^x wasinhibited in the presence of ATP, CTP and UTP, ITP as well asGDP, GTP and dGTP.