The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution

Hie structure of E.coli soluble inorganic pyrophosphatase hasbeen refined at 2.7 resolution to an R-factor of 20.9. Theoverall fold of the molecule is essentially the same as yeastpyrophosphatase, except that yeast pyrophosphatase is longerat both the N- and C-termini. Escherichia coli pyrophosphataseis a mixed +ß protein with a complicated topology.The active site cavity, which is also very similar to the yeastenzyme, is formed by seven ß-strands and an -helixand has a rather asymmetric distribution of charged residues.Our structure-based alignment extends and improves upon earliersequence alignment studies; it shows that probably no more than14, not 15–17 charged and polar residues are part of theconserved enzyme mechanism of pyrophosphatases. Six of theseconserved residues, at the bottom of the active site cavity,form a tight group centred on Asp70 and probably bind the twoessential Mg⁺ ions. The others, more spreadout and more positivelycharged, presumably bind substrate. Escherichia coli pyrophosphatasehas an extra aspartate residue in the active site cavity, whichmay explain why the two enzymes bind divalent cation differently.Based on the structure, we have identified a sequence motifthat seems to occur only in soluble inorganic pyrophosphatases.     

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.     

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.     

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

The expression of immunoglobulin heavy and light chain variableregions in the cytoplasm of Escherichia coli and formation ofa functional heterodimer has been demonstrated. Variable domainsequences were taken from the heavy and light chain cDNAs ofthe monoclonal antibody Gloop 2 and engineered for expressionin a dual origin expression vector. The engineered genes vhg2and vlg2 were separately subcloned into the vector, creatingtwo expression plasmids. Expression of the heavy and light chainvariable region genes (encoding 116 and 109 amino adds respectively)was investigated in eight E.coli strains; the polypeptides wererapidly degraded in a host strain optimized for expression andin E.coli strains deficient in the major protease La (lon^-).Accumulation was permitted in severely protease-deficient E.colihaving a defective heat-shock response. A lon^- mutation in thisgenetic background permitted even higher accumulation. Expressionlevels were 7 and 1% of total bacterial protein for light andheavy chain variable regions respectively. Expression of theheavy chain variable region gene was increased by includinga longer Shine–Dalgarno sequence. Similar constructionsin the light chain vector had no effect on expression levels.The insoluble variable region polypeptides were reconstitutedinto a heterodimer possessing the full antigen binding characteristicsof both the parent monoclonal antibody and its Fab fragment.     

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.     

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

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

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.     

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

The alcohol dehydrogenase gene from the thermophilic archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.     

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.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

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.     

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.     

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa

The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala^*Ala^*Lys^*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala^*Ala^*Lys^*Pro or Lys^*Lys^*Ser^*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)_nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)_nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)_nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)_n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 10⁶ M^–1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)₈OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)_n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)₅AAKTA]_n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other protein–DNA interactions.     

The influence of Glu44 and Glu56 of cytochrome b5 on the protein structure and interaction with cytochrome c

The gene encoding trypsin-solubilized bovine liver microsomalcytochrome b₅ (82 residues in length) has been mutated, in whichthe codons of Glu44 and Glu56 were changed to those of Ala.The mutated genes were expressed in Escherichia coli successfullyand three mutant proteins (E44A, E56A and E44/56A) were obtained.The UV-visible, CD and ¹H NMR spectra of proteins have beenstudied. The results show that the mutagenesis at surface residuesdoes not alter the secondary and tertiary structures of cytochromeb₅ significantly. The interactions between recombinant cytochromeb₅ and its mutants with cytochrome c were studied by using opticaldifference spectra. The results demonstrated that both Glu44and Glu56 of cytochrome b₅ participate in the formation of acomplex between cytochrome b₅ and cytochrome c.     

Overproduction and purification of the regulatory subunit of Escherichia coli aspartate transcarbamoylase

An Escherichia coli strain/plasmid system has been developedfor the overexpression of the regulatory subunit of E.coli aspartatetranscarbamoylase (ATCase). Production of large quantities ofregulatory subunit, by the method described here, should facilitatefuture experiments, such as X-ray crystallography, NMR and hybridizationexperiments, aimed at understanding the heterotropic mechanismthat regulates the activity of ATCase. The plasmid used forthe over-expression carries the gene for the regulatory subunit,pyrI, downstream from the strong pyrB promoter. The host strain,EK1104 [Nowlan, S.F. and Kantrowitz, E.R. (1985) J. Biol. Chem.,260, 14712–14716] carries a deletion in the pyrBI regionof the chromosome, as well as a leaky pyrF allele. When thisstrain/plasmid system is grown under limiting pyrimidine levels,large quantities of the regulatory subunit of ATCase are producedwithout any trace of catalytic subunit or holo-enzyme. A procedurefor the purification of the regulatory subunit from cell extractshas also been developed yielding {small tilde}50 mg of purifiedregulatory subunit per liter of initial culture. The regulatorysubunit produced in this fashion is fully competent in reassociationexperiments with the native catalytic subunit. Furthermore,the reassociated holoenzyme exhibits kinetic properties identicalto those of the wild type enzyme. In addition, we report theconstruction of a pUC119 based plasmid which carries a uniqueNdeI site at the fMet of the pyrB gene of ATCase. This plasmid,which was used in the construction of the system for the overexpressionof the regulatory subunit of ATCase, has been shown to be ofgeneral use for the expression of foreign proteins in E.coli.     

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

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

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.     

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

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