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Biomedical subjects

H Malke

Publications and source records attributed to H Malke.

At least 19 recordsLinked to original sources
PubMed1991

Protein A-streptokinase fusion protein for immunodetection of specific IgG antibodies.

The streptococcal streptokinase gene truncated at its 5' end was fused to regions of the staphylococcal protein A gene encoding the Fc-binding domains A and B. The resultant fusion gene, when expressed in the Escherichia coli lacPO system or under the speA expression/secretion signals in S. sanguis, specified a bifunctional hybrid protein, SPA-SKC, capable of Fc binding and plasminogen activation. When used in immunoassays designed to titrate antisera raised against bovine chymosin, human serum albumin and fibrinogen, the assay using SPA-SKC compared well with that using a commercial SPA-enzyme conjugate. The simple preparative method together with its efficacy and ease of use, make SPA-SKC a potentially valuable detector reagent in quantitative immunology.

Antibodies, Anti-Idiotypic↗
PubMed1990

Duplication of the streptokinase gene in the chromosome of Streptococcus equisimilis H46A.

The erythromycin resistance plasmid pSM752 carrying the cloned streptokinase gene, skc, was introduced by protoplast transformation into Streptococcus equisimilis H46A from which skc was originally cloned. Cells transiently supporting the replication of pSM752 gave rise to an erythromycin-resistant clone designated H46SM which was plasmid free and produced streptokinase at levels approximately twice as high as the wild type. Southern hybridization of total cell DNA with an skc-containing probe provided evidence for the duplication of the skc gene in the H46SM chromosome. The results, which have some bearing on industrial streptokinase production, can be best explained by a single cross-over event between the chromosome and the plasmid in the region of shared homology leading to the integration of pSM752 in a Campbell-like manner.

Blotting, Southern↗
PubMed1989

Sequence-directed DNA bending upstream of the streptokinase promoter.

A 450-base pair (bp) HinfI restriction fragment from the chromosome of Streptococcus equisimilis H46A contains the early coding region of the streptokinase gene (skc), the skc promoter, and a stretch of DNA 5' to the--35 region of the skc promoter. Two-dimensional polyacrylamide (PA) gel electrophoresis at two different temperatures showed that this fragment migrates anomalously slowly on PA gels, suggesting the existence of a bent DNA conformation. Inspection of the nucleotide sequence confirmed this suggestion by revealing numerous oligomeric dA.dT tracts, some of which are in phase with the helix screw. Computer analysis of the sequence predicted the existence of two bending loci, one of which is located upstream of the skc promoter. In addition to showing DNA bending, the 450-bp HinfI fragment contains multiple 13-bp sequences homologous to the Escherichia coli integration host factor DNA-binding consensus sequence. Insertion of IS1 into a site immediately upstream of the--35 region decreased the expression level of skc in E. coli, suggesting that DNA conformation upstream of the promoter has a role in skc expression.

Base Sequence↗
PubMed1989

Novel shuttle vectors for improved streptokinase expression in streptococci and bacterial L-forms.

Novel shuttle vectors of small size and increased copy number capable of replication in Escherichia coli, L-forms of Proteus mirabilis, and streptococci were constructed from a streptococcal erythromycin-resistant plasmid and an Escherichia coli phasmid. The streptokinase gene, skc, was inserted into one of them, and skc expression was studied in Streptococcus sanguis, Streptococcus lactis, and in an L-form strain (LVI) of Proteus mirabilis. The new streptokinase shuttle plasmid, pMLS10 (7.3 kb), specified higher Skc yields in all hosts when compared to pSM752 constructed previously. In particular Proteus mirabilis LVI(pMLS10) proved to be the most productive host, exhibiting complete secretion of the active protein at yields as high as 24000 unit per ml.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Heterologous signal peptide processing in fusion interferon synthesis by engineered L-forms of Proteus mirabilis.

A recombinant DNA Proteus mirabilis L-form expression system, LVI (pJS127), was used to synthesize human fusion interferon alpha 1 (f-IFN-alpha 1). In the expression plasmid used, the complete coding sequence of IFN-alpha 1 was linked to the streptococcal speA promoter and the 5' end of the speA structural gene including its signal sequence coding region. LVI (pJS127) was capable of complete secretion into the culture medium of biologically active f-IFN-alpha 1 whose identity was confirmed by immunological and chemical evidence. In particular, bacterial L-forms were for the first time shown to be capable of correct signal peptide processing, as determined by N-terminal sequencing of the secreted f-IFN.

Amino Acids↗
PubMed1989

The streptokinase gene of group A streptococci: cloning, expression in Escherichia coli, and sequence analysis.

The gene specifying the group A streptokinase (ska) gene was cloned from an M type 49 strain of Streptococcus pyogenes and shown to express in Escherichia coli. The nucleotide sequence of the DNA fragment carrying ska was determined and compared to that of the group C streptokinase gene (skc). There is 90% sequence identity between the two genes, with highly conserved transcription and translation control regions. The deduced amino acid sequence of the group A streptokinase (SKA) contains the same number of amino acids as that of group C streptokinase, with 85% sequence identity between the two proteins. Among 440 amino acid residues specified by the coding sequence, there are 62 non-identical residues with 45 conserved and 17 non-conserved residues. The non-identical residues are located in two major regions, spanning residues 174 to 244 and 270 to 290, with 40 and 10 amino acid changes, respectively. The sequence differences provide an explanation at the molecular level for the previous findings of immunological and chemical heterogeneity among streptokinase produced by pathogenic streptococci.

Amino Acid Sequence↗
PubMed1989

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.

Animals↗
PubMed1989

Heterogeneity of the streptokinase gene in group A streptococci.

A molecular epidemiological study was conducted to determine the distribution of the streptokinase gene in group A streptococcal strains of different M types and in other streptococcal species. Plasmid pNC1, containing only the internal coding sequence of the streptokinase gene from group C streptococcal strain H46A, was used as a DNA probe in colony and Southern hybridization experiments. Only the pathogenic group A, C, and G streptococci contained a streptokinase gene; 12 other Lancefield group strains did not. A total of 134 group A strains, including 61 M types and 6 T types, were tested. Although only 62% (83 of 134) of the strains tested showed positive streptokinase activity by the casein-plasminogen overlay assay, all strains contained the streptokinase gene as evidenced by strong hybridization with the pNC1 probe. Southern blot DNA hybridizations were carried out with 101 strains of group A streptococci. The restriction enzymes HindIII and HaeIII were used to digest the genomic DNA. Six hybridization patterns were observed after HindIII digestion. Double hybridization bands appeared in all of the patterns, which indicated the existence of a highly conserved HindIII site. More complex hybridization results were obtained after HaeIII digestion. Twelve hybridization patterns were observed; three were characterized by a single hybridization band, and nine were characterized by double bands. Variations in hybridization patterns were observed in strains of both the same and different serotypes. The overall results at the gene level indicate that there is considerable heterogeneity among the streptokinases of group A streptococci, consistent with previous findings of immunological and chemical differences among streptokinases of group A streptococci.

DNA Probes↗
PubMed1989

Streptokinase mutations relieving Escherichia coli K-12 (prlA4) of detriments caused by the wild-type skc gene.

A novel phenotype is described for Escherichia coli K-12 carrying the prlA4 allele determining a membrane component of the protein export mechanism. It is manifest as transformation deficiency for plasmids containing the cloned group C streptococcal streptokinase gene, skc. Streptokinase plasmid mutations relieving the prlA4 strain of this deficiency fell into three classes. Class 1 included skc::IS5 insertions, with IS5 integrated in a region encoding the Skc signal sequence and inactivating skc. Class 2 included IS1 insertions leaving skc intact but reducing skc expression, presumably by altering the function of the skc promoter as judged by an insertion site close to the -35 region. The most interesting class, 3, included skc deletions removing the entire signal sequence or a tetrapeptide from its hydrophobic core. The tetrapeptide deletion reduced the size, hydrophobicity, and predicted alpha-helicity of the central region of the Skc signal sequence but facilitated the export of mature Skc in both the wild type and the prlA4 mutant. These findings indicate that the incompatibility between prlA4 and skc is related to deleterious effects of the Skc signal sequence. The tetrapeptide deletion may function by altering the conformation of the signal sequence so as to render interaction with both the PrlA wild-type protein and the PrlA4 mutant protein less detrimental to the export mechanism. These findings also provide an explanation for the difficulties encountered in cloning streptokinase genes in E. coli plasmids and maintaining their structural stability.

DNA Mutational Analysis↗
PubMed1988

Expression of the human interferon-alpha 1 gene under transcriptional and translational control of the speA gene.

The human gene for mature interferon-alpha 1 (IFN-alpha 1) was inserted in a new transcription-translation fusion vector system based on the expression and secretion signals of the gene for type A streptococcal pyrogenic exotoxin, speA. As deduced from the known nucleotide sequences of the component elements, the encoded IFN-alpha 1 was a fusion protein carrying an N-terminal extension of 17 amino acids. When inserted in appropriate vectors capable of replication in Escherichia coli, Bacillus subtilis and Streptococcus sanguis, this expression configuration directed the synthesis of antiviral activity in all 3 organisms, as judged by the cythopathic effect inhibition assay of Vesicular Stomatitis Virus. In E. coli JM101, IFN activity was found mainly in the cytoplasmic protein fraction whereas in the gram-positive hosts, it was completely secreted into the culture medium.

Amino Acid Sequence↗
PubMed1988

Survey of the pSM10 family of streptococcal cloning vectors.

A survey of the pSM10 family of streptococcal plasmid cloning vectors is provided. These vectors are derived from a macrolide-lincosamide-streptogramin B resistance plasmid and include general cloning vectors, shuttle vectors, helper plasmids, expression vectors, gene fusion vectors and plasmids. Besides describing their general properties, this paper also provides their genetic and physical maps.

Cloning, Molecular↗
PubMed1988

Tripartite streptokinase gene fusion vectors for gram-positive and gram-negative procaryotes.

A specific 1,596 bp HincII fragment ('skc) from the chromosome of Streptococcus equisimilis contains an active streptokinase (SK) gene (skc) lacking, in addition to the expression signals, codons 1 through 39 of wild-type skc but retaining the remainder of the skc coding sequence together with the transcription terminator. Using this fragment as an indicator gene, we constructed two types of vectors which in appropriate hosts resulted in the synthesis of SK fusion proteins after insertional activation of 'skc. The first type are open reading frame (ORF) vectors in which 'skc was inserted into pUC18 out of frame with respect to lacZ', thus conferring an SK-negative phenotype. Any DNA fragments representing ORFs inserted between the lacZ' expression signals and 'skc such that the skc reading frame was restored resulted in the production of tripartite proteins which exhibited SK activity. The second type of vector, which functioned in both gram-positive and gram-negative bacteria, used the streptococcal speA expression and secretion signals in front of the ORF to activate 'skc insertionally. Using a large fragment from the chymosin gene as the target sequence, the usefulness of these vectors for studying foreign gene expression in streptococci as well as Escherichia coli was demonstrated.

Amino Acid Sequence↗