Synthesis and expression in Escherichia coli of the gene for the albumin-binding domain of Streptococcus protein G]

The chemical-enzymatic synthesis of a gene coding for A2B2 repeats of the albumin-binding domain of streptococcal protein G has been accomplished. The codon usage of the natural gene has been modified to adapt an artificial sequence for the efficient translation in E. coli. The gene (238 b.p.) was cloned in the polylinker plasmid pUCL1 and then fused in frame to the 3'-terminus of the gene for the IgG-binding domain of staphylococcal protein A, which was earlier cloned in the expression plasmid pUCL2. A fused polypeptide composed of the E and B domains of protein A and A2B2 repeats of protein G was produced in E. coli cells under the lac promoter control. The resulted product was isolated by affinity chromatography on IgG-sepharose and (or) albumin-sepharose.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus.

The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.     

Some improved versions of methods for the indirect detection of staphylococcal protein A gene expressed in Escherichia coli.

Several versions of methods for the indirect detection of expression of staphylococcal protein A gene (spa) in Escherichia coli (E. coli) were devised by making use of biological properties of staphylococcal protein A (SpA). i) Hemagglutination of sheep red blood cells (SRBC) sensitized with anti-SRBC-antibodies using heat-treated spa-transformed E. coli organisms; Native spa-transformed E. coli organisms did not agglutinate the sensitized SRBC. The heat-treatment (60 C, 4 hr) of the transformants, however, caused positive hemagglutination like SpA-positive Staphylococcus aureus (S. aureus) organisms. ii) Halo formation around colonies on agar plates containing normal dog serum, which is originally used for the detection of SpA of S. aureus. A mutant strain NMJ was isolated, which showed formation of the halo of precipitate due to interaction between immunoglobulin and SpA. iii) A new version of immunodetection; After lysis of the transformants grown on a nitrocellulose membrane by alkali, SpA could be directly detected by immuno-detection procedures after inactivation of endogenous peroxidase in bacteria by phenylhydrazine and hydrogen peroxide.     

Enzyme immunoassay system for detection of staphylococcal enterotoxin,type C

An enzyme immunoassay (EIA) system for detection of staphylococcal enterotoxin, type C, has been developed. The sensitivity of the system is 1 ng/ml. The optimum EIA parameters have been worked out. The absence of false positive results with heterologous toxins confirms the specificity of the assay system. The possibility of the detection of staphylococcal enterotoxin, type C, in staphylococci isolated from different sources has been shown.     

Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin

Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.     

Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images     

The comparative characteristics of the immunomodulating properties of protein A from Staphylococcus aureus of different origins

The immunomodulating properties of highly purified staphylococcal protein A and its analog obtained by gene engineering techniques have been compared with those of commercial preparations. The comparison has shown that the differences observed in this investigation may be explained by the presence of admixtures of staphylococcal nature in commercial preparations. The preparations of highly purified staphylococcal and recombinant protein A stimulate humoral immune response and the processes of phagocytosis and do not show mitogenic activity with respect to T cells. The conclusion on the identity of the immunomodulating activity of the preparations of natural and recombinant protein A has been made.     

Synthesis and expression of an artificial gene of an IgG-binding fragment of protein A from Staphylococcus aureus

The chemical-enzymatic synthesis of a gene for IgG-binding fragment of the staphylococcal protein A has been carried out. The design of the gene, which consists of signal peptide and modified E and B domains, and strategy of the synthesis provided possibility of various degrees of polymerization of the gene fragment coding for B domain and of the whole gene. Several protein A-like polypeptides composed of the leader sequence, E domain and 1 to 4 copies of B domain were produced in E. coli cells under the lac promoter control.     

Comparative structural analysis of staphylococcal enterotoxins A and E

Structural analysis of staphylococcal enterotoxins A and E, two functionally and serologically related proteins, has been carried out using circular dichroism, and tryptophan fluorescence quantum yield and quenching. Secondary structures derived from the far-UV circular dichroic spectra revealed that both enterotoxins are in predominantly beta-sheets/beta-turn structures (80-85%). Staphylococcal enterotoxin A has significantly higher alpha-helical content (10.0%) than staphylococcal enterotoxin E (6.5%). Tryptophan fluorescence spectra of both enterotoxins showed maxima at approximately 342 nm, indicating that the fluorescent tryptophan residues are in polar environments. However, the tryptophan fluorescence quantum yields indicated that tryptophan residues are approximately 41% more fluorescent in staphylococcal enterotoxin A than in staphylococcal enterotoxin E. Tryptophan fluorescence quenching by a surface quencher, I-, and a neutral quencher, acrylamide, indicated that at least 1 of the 2 tryptophan residues in both staphylococcal enterotoxins A and E is located on the outer surface of the proteins. This tryptophan residue is in significantly different environments in the two enterotoxins. Six antigenic sites are predicted from the hydrophilicity and secondary structure information; at least four sites are identical. In general, staphylococcal enterotoxins A and E have some structural similarities which are compatible with their common biological activities.     

Variability of the pathogenicity traits of populations of staphylococci in a surgical hospital

The results of the study of heterogeneity of staphylococcal populations at a surgical ward are presented. The study deals with qualitative and quantitative characteristics of three groups of pathogenicity factors: protease (the penetration factor), protein A (the function of protection from phagocytosis) and alpha-hemolysin (the toxic function). The study shows that the greatest number of S. aureus strains with a high content of protein A has been isolated from patients with postoperative and wound infections. On the basis of the data obtained in this study the groups of strains have been defined in accordance with the association of the signs of pathogenicity. These groups reflect pronounced heterogeneity of staphylococcal strains at a surgical ward.     

Enterotoxigenic capacity of strains of Klebsiella and Enterobacter genera isolated in acute intestinal diseases in children

234 strains, including 104 K. pneumoniae strains, 28 K. oxytoxica strains, 64 E. cloacae strains and 40 E. aerogenes strains, have been isolated from the intestine of 266 children with diarrhea, aged up to 1 year, and studied for enterotoxigenicity. By the coagglutination test, made with G. Kronvall's staphylococcal reagent prepared with the use of antiserum to Escherichia coli LT-enterotoxin, and the biological assay on suckling mice enterotoxigenic activity has been revealed in 119 strains, including 48 K. pneumoniae strains (12.6%), 33 E. cloacae strains (27.4%) and 23 E. aerogenes strains (19.7%). The strains producing only LT-enterotoxins, only ST-enterotoxins, and both LT- and ST-enterotoxins have been found. The determination of the enterotoxigenic activity of the clinical isolates of opportunistic enterobacteria makes it possible to improve the etiological interpretation of acute intestinal infections.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E.

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Choice of the method for isolating conjugates of staphylococcal protein A with peroxidase for immunoenzyme analysis

An attempt to obtain the preparations of peroxidase-labeled staphylococcal protein A, intended for use in the enzyme immunoassay, by the glutaraldehyde method has failed. The modified periodate method permitting the preparation of active conjugates of staphylococcal protein A with peroxidase has been developed.     

Structure of the IgG-binding regions of streptococcal protein G.

The gene encoding the IgG-binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5-kb insert gave a functional product in Escherichia coli. Protein analysis of affinity-purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG-binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N-terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C-terminal part of streptococcal protein G consists of three IgG-binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.     

Detection of staphylococcal enterotoxins A, B, C, D, and E in foods by radioimnunoassay, using staphyloccal cells containing protein A as immunoadsorbent.

A sensitive radioimmunoassay utilizing Staphylococcus aureus cells containing protein A as a coprecipitant was developed for the detection and quantitation of staphylococcal enterotoxins A, B, C, D, and E in a variety of foods. The enterotoxins were extracted from the foods by a simple and rapid procedure. The sensitivity of the assay is 1.0 ng or less of enterotoxin per g of food.     

Staphylococcal enterotoxin type D: isolation, purification, identification

The three step scheme of isolation and purification of staphylococcal type D enterotoxin was elaborated to obtain the homogeneous protein. The scheme includes protein concentration by (NH4)2SO4 saturation and subsequent fractionation on DEAE-cellulose and Sephacryl S-200. The yield of homogeneous protein is 25.5%. The molecular mass of the 29,600 D protein was identified by electrophoresis in polyacrylamide gel in the presence of SDS. The enterotoxic dose for staphylococcal type D enterotoxin is 5 mkg per kg of body mass as identified in experiments with cats. Immunological identity of the obtained protein was established to the commercial preparation of staphylococcal enterotoxin D produced by "Serva".     

The human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.