High-level expression of a biologically active staphylokinase in Pichia pastoris.
Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310 mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20-37°C and pH ranges of 6.8-9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042 U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720 µg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6 kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.
a Department of Medical Biotechnology, Faculty of Medicine , Mashhad University of Medical Sciences , Mashhad , Iran.
Habibollah
H
Faraji
b Pharmaceutical Research Center, School of Pharmacy , Mashhad University of Medical Sciences , Mashhad , Iran.
Mohammad
M
Ramezani
c Neurocognitive Research Center, Faculty of Medicine , Mashhad University of Medical Sciences , Mashhad , Iran.|d Department of New Sciences and Technology, Faculty of Medicine , Mashhad University of Medical Sciences , Mashhad , Iran.
Hamid Reza
HR
Sadeghnia
e Pharmaceutical Research Center , Mashhad University of Medical Sciences , Mashhad , Iran.|f Department of Medicinal Chemistry , Mashhad University of Medical Sciences , Mashhad , Iran.
Khalil
K
Abnous
g Biotechnology Research Center , Mashhad University of Medical Sciences , Mashhad , Iran.
Fatemeh
F
Soltani
h Department of Medical Biochemistry, Faculty of Medicine , Mashhad University of Medical Sciences , Mashhad , Iran.
Baratali
B
Mashkani
D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D003062:Codon; D003470:Culture Media / Q000378:metabolism; D015870:Gene Expression; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D003062:Codon; D003470:Culture Media; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D000432:Methanol
Codon optimization; Pichia pastoris; high-level expression; methanol; specific activity; staphylokinase