Production, crystallization and diffraction to atomic resolution of an antibody Fv specific for the blood-group A oligosaccharide antigen.

The histoblood-group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single-chain variable-domain antigen-binding fragment (scFv) gene based on the known sequence of an anti-blood-group-A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli. The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms. The corresponding variable-domain antigen-binding fragment (Fv) was generated by cleaving the VL-VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin-A-trisaccharide conjugate (KD = 290 microM). AC1001 Fv crystals grown in the presence of N-acetylgalactosamine diffracted to 0.93 A resolution. This is the first reported example of a crystal of an antibody antigen-binding fragment diffracting to atomic resolution.

Effect of C lambda-C kappa domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate Fabs.

We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti-carbohydrate Fabs, one specific for a Brucella cell-surface polysaccharide and the second for the human blood group A determinant. Very similar VL amino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized VH genes were fused to a previously made sequence encoding the C(gamma 1)1 domain as an alternative to synthesis of the natural C gamma 2b 1 and C mu 1 sequences. The VL genes were initially coupled to a synthetic C kappa gene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the C kappa domains with a C lambda 1 domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The C kappa and C lambda 1 forms of each of the Brucella and blood group A Fabs, with His5 fusions at the C-termini of the Fd chains, were purified by immobilized metal affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues.

The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.

Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.

The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.

Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product.

A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.

Identification of two distinct Bacillus circulans xylanases by molecular cloning of the genes and expression in Escherichia coli.

Two genes coding for xylanase synthesis in Bacillus circulans were cloned and expressed in Escherichia coli. After digestion of genomic DNA from Bacillus circulans with EcoRI and PstI, the fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Restriction enzyme mapping of the two inserts coding for xylanase activity indicated distinctly different nucleotide sequences. Cross-hybridization assays confirmed the absence of sequence homology between the two genes. In vitro transcription-translation assays indicated that the cloned genes encoded for proteins with molecular weights of 22,000 and 59,000. The gene products displayed different substrate specificities. The 22,000-dalton enzyme readily hybrolyzed aspeen, larchwood, and oat spelt xylans, whereas the second was unable to extensively depolymerize oat spelt xylan and resulted in very limited reducing sugar release from any of the xylan substrates tested. Both of the xylanases had isoelectric points of approximately 9.0.

Identification of three distinct Clostridium thermocellum xylanase genes by molecular cloning.

Three genes coding for xylanase synthesis in Clostridium thermocellum were cloned and expressed in Escherichia coli. Genomic DNA from Clostridium thermocellum was digested to completion with HindIII, BamHI, and SalI. The fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Two of the genes encoded for xylanases which depolymerized xylans but were unable to extensively convert these substrates to reducing sugar. The third gene encoded for an enzyme that extensively hydrolyzed xylan. The insert containing the latter gene was subjected to extensive mapping and was found to encode for a xylanase with a molecular weight of approximately 25,000. The protein product of the cloned gene was obtained in a relatively pure form by heat treatment, ion exchange and gel permeation steps. The enzyme was quite stable to high temperatures with a half-life of 24 h at 70 degrees C.

Ferulic Acid Esterase Activity from Schizophyllum commune.

Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate.

Molecular Cloning and Expression of a Xylanase Gene from Bacillus polymyxa in Escherichia coli.

Genomic fragments of Bacillus polymyxa derived from separate and complete digestion by EcoRI, HindIII, and BamHI were ligated into the corresponding sites of pBR322, and the resulting chimeric plasmids were transformed into Escherichia coli. Of 6,000 transformants screened, 1 (pBPX-277) produced a clear halo on Remazol brilliant blue xylan plates. The insert in the pBPX-277 recombinant, identified as an 8.0-kilobase BamHI fragment of B. polymyxa, was subsequently subjected to extensive mapping and a series of subclonings into pUC19. A 2.9-kilobase BamHI-EcoRI subfragment was found to code for xylanase activity. Xylanase activity expressed by E. coli harboring the cloned gene was located primarily in the periplasm and corresponded to one of two distinct xylanases produced by B. polymyxa. Xylanase expression by the cloned gene occurred in the absence of xylan and was reduced by glucose and xylose. Southern blot hybridization with the cloned fragment as a probe against complete genomic digests of the bacilli B. polymyxa, B. circulans, and B. subtilis revealed that the cloned xylanase gene was unique to B. polymyxa. The xylanase expressed by the cloned gene had a molecular weight of approximately 48,000 and an isoelectric point of 4.9.

Induction of Cellulolytic and Xylanolytic Enzyme Systems in Streptomyces spp.

Extracellular enzyme preparations from Streptomyces flavogriseus and Streptomyces olivochromogenes cultures grown on cellulose contained primarily cellulase activities, but similar preparations from cultures grown on xylan-containing materials possessed high levels of both cellulase and xylanase activities. Growth conditions that gave high endoxylanase levels also resulted in the production of enzymes involved in the hydrolysis of the nonxylose components of xylan. Specific acetyl xylan esterase activities were identified in enzyme preparations from both organisms. Both organisms also produced alpha-l-arabinofuranosidase activity that was not associated with endoxylanase activity. Other activities produced were alpha-l-O-methylglucuronidase and ferulic acid esterase. The latter enzyme was produced only by S. olivochromogenes and is an activity which has not previously been identified as a component of hemicellulase preparations.

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus.

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.

Studies on Cellulose Hydrolysis by Acetivibrio cellulolyticus.

Acetivibrio cellulolyticus extracellular cellulase extensively hydrolyzed crystalline celluloses such as Avicel (FMC Corp., Food and Pharmaceutical Products Div., Philadelphia, Pa.) but only if it was desalted and supplemented with Ca. The Ca effect was one of increased enzyme stability in the presence of the ion. Although preincubation of the cellulase complex at 40 degrees C for 5 h without added Ca had a negligible effect on endoglucanase activity or on the subseqent hydrolysis of amorphous cellulose, the capacity of the enzyme to hydrolyze crystalline cellulose was almost completely lost. Adsorption studies showed that 90% of the Avicel-solubilizing component of the total enzyme preparation bound to 2% Avicel at 40 degrees C. Under these conditions, only 15% of the endoglucanase and 25% of the protein present in the enzyme preparation adsorbed to the substrate. The protein profile of the bound enzyme, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was complex and distinctly different from the profile observed for total cellulase preparations. The specific activity of A. cellulolyticus cellulase with respect to Avicel hydrolysis was compared with that of commercially available Trichoderma reesei cellulase.

Purification and characterization of an exoglucanase from Streptomyces flavogriseus.

Streptomyces flavogriseus, a mesophilic actinomycete, produces high levels of extracellular enzymes capable of hydrolyzing cellulose and xylan. One such enzyme, an exoglucanase, has been purified to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel permeation chromatography on Bio-Gel P-60, preparative isoelectric focusing, and concanavalin A affinity chromatography. This purification sequence disclosed the presence of several distinct endoglucanase and xylanase fractions. Homogeneity of the purified enzyme was demonstrated by analytical isoelectric focusing and sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 45 000 and an isoelectric point of 4.15. The enzyme demonstrated negligible activity with carboxymethylcellulose as the substrate. It was able to extensively hydrolyse acid-swollen cellulose; the main product of enzyme action was cellobiose.

Streptomyces flavogriseus cellulase: Evaluation under various hydrolysis conditions.

Streptomyces flavogriseus CMCase and Avicelase were very stable at 30 degrees C but not at 40 degrees C or higher. beta-Glucosidase was less stable at all temperatures tested. Stabilities were similar at pH values between 5.5 and 7, the optimal range for enzyme activity. Cellulose solubilizing activity was reduced by 40% at a cellobiose concentration of 150 mM but glucose inhibited activity by only 10% at this concentration. beta-Glucosidase was inhibited by 40% at a glucose concentration of 10mM (ten times the substrate concentration). Relatively dilute S. flavogriseus cellulase extensively hydrolysed acid-swollen cellulose at concentrations as high as 10%. More highly crystalline forms of cellulose were more resistant to attack.