RESUMO
To produce an effective recombinant streptokinase [rSK] from pathogenic Streptococcus pyogenes isolate in yeast, and evaluate its potential for thrombolytic therapy. This study was conducted from November 2012 to December 2013 at King Khalid University, Abha, Kingdom of Saudi Arabia [KSA]. Throat swabs collected from 45 pharyngitis patients in Asser Central Hospital, Abha, KSA were used to isolate Streptococcus pyogenes. The bacterial DNA was used for amplification of the streptokinase gene [1200 bp]. The gene was cloned and in vitro transcribed in an eukaryotic expression vector that was transformed into yeast Pichia pastoris SMD1168, and the rSK protein was purified and tested for its thrombolytic activity. The Streptococcus pyogenes strain was isolated and its DNA nucleotide sequence revealed similarity to other Streptococcus pyogenes in the Gene bank. Sequencing of the amplified gene based on DNA nucleotide sequence revealed a SK gene closely related to other SK genes in the Gene bank. However, based on deduced amino acids sequence, the gene formed a separate cluster different from clusters formed by other examined genes, suggesting a new bacterial isolate and accordingly a new gene. The purified protein showed 82% clot lysis compared to a commercial SK [81%] at an enzyme concentration of 2000 U/ml. The present yeast rSK showed similar thrombolytic activity in vitro as that of a commercial SK, suggesting its potential for thrombolytic therapy and large scale production.
RESUMO
Alkaline phosphatase was purified to homogeneity from Pseudomonas aeruginosa 50071 starting with heat treatment at 60°C in the presence of Co[2]+ and ammonium sulphate fractionation, followed by separation on DEAE-Sephadex A50 and G-100 Sephadex gel filtration. The enzyme was purified 468 fold and showed a final specific activity of 51.5 unit/mg protein with a yield 42%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE] of the purified enzyme revealed a single protein band of molecular weight 82 KDa. The molecular weight of the native enzyme was calculated to be 158 KDa, as determined by gel filtration, which approximated to two subunits together [164 KDa] suggesting a homogeneous dimeric structure of the enzyme. The enzyme showed a maximum activity at pH 9.5 when incubated at 37°C. A Lineweaver-Burk analysis gave a K[m] of 2.1 mM and V[max] of 8.0 U/ml. The enzyme activity was enhanced by addition of CoCl[2] and ZnCl[2] together. The amino acid composition of the purified enzyme was also determined