Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system.

Streptokinase is one of the major blood-clot-dissolving agents used in many medical treatments. With the cloned streptokinase gene (skc) available, production of the secreted streptokinase from various Bacillus subtilis strains was studied. The use of the six-extracellular-protease-deficient strain, WB600, greatly improved the production yield of the secreted streptokinase. A modified skc which has the original skc promoter and signal sequence replaced with the B. subtilis levansucrase promoter and signal sequence was also constructed. B. subtilis carrying either the wild-type or the modified skc produces streptokinase at a comparable level. Even with WB600 as the expression host, a C-terminally-processed streptokinase was also observed. Through region-specific combinatorial mutagenesis around the C-terminal processing sites, streptokinase derivatives resistant to C-terminal degradation were engineered. One of the derivatives showed a 2.5-fold increase in specific activity and would potentially be a better thrombolytic agent.     

Constitutive expression of Pasteurella multocida toxin

Abstract The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of β-galactosidase from a cps-lacZ fusion ( lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.     

The streptokinase gene

The subject of this paper is the molecular cloning, nucleotide sequencing, and expression in heterologous hosts of the streptokinase gene (skc) from the group C streptococcal strain H46A. The skc gene shows no extended regions homologous to the staphylokinase gene.     

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.     

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.     

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.     

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.     

The organization of the prosystemin gene

The organization of the gene encoding tomato prosystemin, a 200 amino acid protein precursor of the 18 amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato plants, is reported. The prosystemin sequence reveals that the gene, which is composed of five homologous pairs of exons plus a non-homologous exon at the C-terminus containing the systemin sequence, has evolved by several gene duplication-elongation events from a much smaller ancestral gene. The nucleotide and amino acid sequence homologies among the exons suggest that a small ancestral gene was duplicated to form at least two tandem repeats, followed by subsequent duplication-elongation events that resulted in five tandemly repeated nucleotide sequences and three duplicated amino acid sequence elements. Since the systemin nucleotide or amino acid sequence was not duplicated, it was either not part of the gene duplication-elongation events or its coding region evolved separately and may even have been added to the tandemly repeated part of the gene at a later time.     

Complete amino acid sequence of streptokinase and its homology with serine proteases

The complete amino acid sequence of streptokinase has been determined by automated Edman degradation of its cyanogen bromide and proteolytic fragments. The protein consists of 415 amino acid residues. Sequence microheterogeneity was found at two positions. The NH2-terminal 245 residues of streptokinase are homologous to the sequences of several serine proteases including bovine trypsin and Streptomyces griseus proteases A and B. The sequence alignment suggests that the active-site histidine-57 has changed to a glycine in streptokinase. The other active-site residues, aspartyl-102 and serine-195, are, however, present at the expected positions. Streptokinase also contains internal sequence homology between the NH2-terminal 173 residues and a COOH-terminal 162-residue region between residues 254 and 415. Moderate homology in predicted secondary structures also exists between these two regions. Although streptokinase is not a protease, these observations suggest that it has evolved from a serine protease by gene duplication and fusion. A COOH-terminal region of about 80 residues is apparently deleted from the second half of the duplicated structures. These observations further suggest that the three-dimensional structure of streptokinase likely contains two independently folded domains, each homologous to serine proteases.     

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.     

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.     

Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae.

We have cloned and expressed in Escherichia coli a gene encoding a 15,000-apparent-molecular-weight peptidoglycan-associated outer membrane lipoprotein (PAL) of Haemophilus influenzae. The nucleotide sequence of this gene encodes an open reading frame of 153 codons with a predicted mature protein of 134 amino acids. The amino acid composition and sequence of the predicted mature protein agree with the chemically determined composition and partial amino acid sequence of PAL purified from H. influenzae outer membranes. We have also identified a second gene from H. influenzae that encodes a second 15,000-apparent-molecular-weight protein which is recognized by antiserum against PAL. This protein has been shown to be a lipoprotein. The nucleotide sequence of this gene encodes an open reading frame of 154 codons with a predicted mature protein of 136 amino acids and has limited sequence homology with that of the gene encoding PAL. Southern hybridization analysis indicates that both genes exist as single copies in H. influenzae chromosomal DNA. Both genes encode polypeptides which have amino-terminal sequences similar to those of reported membrane signal peptides and are associated primarily with the outer membrane when expressed in E. coli.     

Identification by a random sequencing strategy of the fowlpoxvirus DNA polymerase gene, its nucleotide sequence and comparison with other viral DNA polymerases.

The nucleotide sequence of the DNA polymerase gene of the avipoxvirus fowlpox is presented and the predicted amino acid sequence compared with that of the orthopoxvirus vaccinia. The results have brought to light an error in the vaccinia sequence which has resulted in the ommission of 44 amino acids from the carboxy-terminus of the vaccinia DNA polymerase. There has been extensive conservation of amino acids throughout the enzymes, and regions identified as being present in DNA polymerases from a wide range of viruses are again present here. The method used to identify the fowlpoxvirus gene could have applications towards defining genomic organisations in other viral systems.     

Active streptokinase from the cloned gene in Streptococcus sanguis is without the carboxyl-terminal 32 residues

The streptokinase expressed by the cloned gene in Streptococcus sanguis has a molecular weight of about 44 000 [Malke, H., Gerlach, D., Kohler, W., & Ferretti, J.J. (1984) MGG, Mol. Gen. Genet. 196, 360-365] while the molecular weight of the native streptokinase is 47 000. The structural and activity differences of the cloned streptokinase (cSK) as expressed by S. sanguis and the native streptokinase (nSK) were investigated. From a partially purified cSK, two active fractions were obtained by reversed-phase HPLC. The minor fraction cSKL was nearly as active as SK in plasminogen activation. The major fraction cSKs had only about one-fourth of the specific activity. The structures of cSKL and cSKs were studied and compared to the known amino acid sequence of SK [Jackson, K. W., & Tang, J. (1982) Biochemistry 21, 6620-6625]. From the NH2- and COOH-terminal sequences and amino acid composition of the cyanogen bromide (CNBr) fragments, it could be deduced that cSKL and cSKs are without 31 and 32 residues, respectively, from the COOH-terminal end of SK. Since the cloned gene contained the full SK structure, the missing structures must have been due to posttranslational proteolysis. An SK fragment similar in size to cSK was observed from a chymotryptic digest of SK.     

蝮蛇毒碱性磷脂酶A_2基因的克隆

从蝮蛇毒腺中抽提总ＲＮＡ．利用人工合成寡核苷酸引物作逆转录,以ｃＤＮＡ为模板进行体外扩增,获得磷脂酶Ａ２（简称ＰＬＡ２）基因,克隆至ｐＢＳ－ｋｓ载体中。通过对３个碱性ＰＬＡ２（简称ＢＰＬＡ２）基因单独克隆分别作ＤＮＡ全序列分析,推导ｐｒｏ－ＢＰＬＡ２由１３８个氨基酸残基构成,与已测定的部分氨基酸序列比较,基本相符。该基因成功的克隆,不仅推导出ＢＰＬＡ２的蛋白质全序列,也为进一步开展蛇毒功能肽蛋白质工程的研究工作打下了良好的基础。