Skc Gene Research Articles

Analysis of genomic region spanning Saltol using SSR markers in rice genotypes showing differential seedlings stage salt tolerance

Micro satellite markers located in the Saltol QTL of 5 Mb region (10.4–15.6 Mb) in chromosome 1 confering seedling stage salt tolerance were used to evaluate 94 rice genotypes. Out of 21, eight SSR markers at Saltol region of Chromosome were found polymorphic. Based on the phenotypic screening, 94 genotypes were grouped as highly tolerant (20), tolerant (18) moderately tolerant (32), sensitive (19) and highly sensitive (5). The marker RM3412 appears to be diagnostic of salinity tolerance and associate to salinity tolerance at seedling stage as it is closely linked to SKC gene. Based on Saltol markers study, CSR 31, CSR 38, CSR 41, CSR 32, Wild 11, CSR 18, Azgo, Pant Dhan 4, Trichi 1, CSR 10 and IR64426-4B-11-1 could not be identified as tolerant genotypes though had expressed tolerant to highly tolerant phenotype to salinity stress at seedling stage, suggesting that QTLs other than Saltol might be controlling their salinity tolerance. It is suggested that these genotypes could serve as potentially novel germplasm and could be exploited for the development of new breeding lines with high level of salinity tolerance by pyramiding of the Saltol and other QTLs.

Heterologous production of streptokinase as a secretary form in Streptomyces lividans and nonsecretary form in Escherichia coli

The skc gene encoding streptokinase (SK), with a molecular weight of approximately 47.4 kDa, was cloned from Streptococcus eouisimilis ATCC9542 and heterologously overexpressed in Streptomyces lividans TK24 and E. coli using various strong promoters. When the sprT promoter was used in the S. lividans TK24 host, the SK protein corresponding to 47.4 kDa was detected with a smaller hydrolyzed protein (44 kDa), implying posttranslational hydrolysis occurred as reported in other expression systems. Casein/plasminogen plate assay revealed that plasmid construct with the signal peptide of SK was superior to that with the signal peptide of sprT in SK production. The maximum productivity of SK was calculated as less than 0.25 unit/ml of the culture broth, which was similar level to those from other expression systems hiring ermE and tipA promoters in the same host. When the skc gene was expressed in E. coli BL21(DE3)pLys under control of T7 promoter, relatively large amount of SK was expressed in soluble form without hydrolyzed protein. The SK activity produced by E. coli/pET28a-T7pSKm was more than 2 units/ml of culture even though about half of the expressed protein formed inactive inclusion body.

Rare codon priority and its position specificity at the 5′ of the gene modulates heterologous protein expression in Escherichia coli

Rare codons and their effects in heterologous protein expression in Escherichia coli were addressed by many investigators. Here, we propose that not all rare codons of a foreign gene have negative effect but selective codon among them and its specific position in the downstream of the start codon modulates the expression. In our study, streptokinase (47 kDa), encoded by skc gene of Streptococcus equisimilis was expressed in E.coli. The analysis of relative codon frequency of skc gene in E.coli reveals the presence of 30% of rare codons in it. Nevertheless, E.coli managed to yield over-expression of this target protein. To explore the codon bias in expression, we have introduced the selective AGG codon at different positions of skc gene such as +2,+3,+5,+8,+9 and +11. The results revealed that at +2 position “AGG” aided over-expression while shifting to +3 and +5 positions it rendered nil expression. In contrary, shifting of AGG codon to later positions like +9 and +11 the inhibitory effect was reversed and resulted in over-expression. The effect of ‘AGG’ rare codon was further studied in GFP expression. In conclusion, besides the choice of rare codons, their precise positions in the foreign gene dictate the level of protein expression.

Constitutive Expression and Optimization of Nutrients for Streptokinase Production by Pichia pastoris Using Statistical Methods

The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett-Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids in Escherichia coli.

A system is described that enables the cloning of genes specifying detrimental proteins in Escherichia coli. The system is based on pUC plasmids and was developed for the expression of the Bacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression of csaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked the csaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of the csaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as the skc gene of Streptococcus equisimilis, which cannot be cloned in high copy numbers in E. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.

Secretion of streptokinase fusion proteins from Escherichia coli cells through the hemolysin transporter

The hemolysin (HlyA) secretion system was used to achieve the sec-independent secretion of streptokinase (Skc) originating from Streptococcus equisimilis into the medium by Escherichia coli cells. The in-frame fusions of the skc gene, either possessing or lacking a region encoding the signal peptide (SP) with the 3′-end of the hlyA gene of various lengths were analysed. All hybrids retained Skc activity. Hybrid proteins devoided of the N-terminal SP, regardless of length of the hlyA secretion signal (62 vs. 194 amino acids), were secreted into the medium by the E. coli HlyA transporter at similar levels. Considerable amounts of hybrid proteins were still, however, associated with E. coli cells, mainly in the degraded form.

Duplication of the streptokinase gene in the chromosome ofStreptococcus equisimilisH46A

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.

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.

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.

Expression of the streptokinase gene from Streptococcus equisimilis in Bacillus subtilis.

The previously cloned and sequenced streptokinase gene (skc) from Streptococcus equisimilis H46A was inserted into plasmid vectors capable of replication in Bacillus subtilis. The skc gene was expressed by use of its own transcription and translation signals which appeared to meet the stringent requirements of B. subtilis for efficient foreign gene expression. The secreted streptokinase activity began to decline toward the end of the exponential growth phase suggesting that B. subtilis exoproteases hydrolyzed and inactivated the foreign protein.

Nucleotide sequence of the streptokinase gene from Streptococcus equisimilis H46A

The entire nucleotide sequence of a cloned 2568-bp PstI fragment from the genome of Streptococcus equisimilis H46A encoding the streptokinase gene ( skc) has been determined. The longest open reading frame comprises 1320 bp which code for streptokinase. The protein is synthesized with a 26-amino acid residue N-terminal extension having properties characteristic of a signal peptide. Comparison of the deduced amino acid sequence with the available amino acid sequence of a commercial streptokinase reveals minor primary structure differences. The nucleotide sequencing of skc does not support the hypothesis that the gene has evolved by duplication and fusion, as suggested by internal twofold amino acid homologies of its product. Furthermore, the skc gene sequence shows no extended regions homologous to the staphylokinase gene. Upstream from the skc gene, the putative skc promoter and the ribosome-binding site sequence have been identified; downstream from the coding region, inverted repeat sequences thought to function as transcription terminators have been detected.