Expression, purification, and activity identification of Alfimeprase in Pichia pastoris.
Alfimeprase (ALF) is a truncated form of non-hemorrhagic zinc metalloproteinase fibrolase. In order to achieve a high level secretion and full activity expression of ALF, the Pichia pastoris (P. pastoris) expression system was used. ALF coding sequence fused with a 6 *histidine tag and an enterokinase recognition site at the N-terminus was cloned into the expression vector pPIC9K and then expressed in P. pastoris strains of GS115 and KM71 by methanol induction. SDS-PAGE and Western blotting analysis showed that the secreted recombinant ALF (rALF) had a molecular weight of 23.8 kDa and was bound specifically to mouse anti-His. tag monoclonal antibody. Under the optimized culture parameters of pH value, initial A(600) value, methanol daily addition concentration and induction time length, the production of rALF reached up to 510 mg/L and 465 mg/L of the GS115 and KM71 transformants, respectively. It also appeared that KM71 was producing a more pure protein than GS115 while GS115 was producing more rALF per unit volume. Through one-step affinity chromatography, the purity of rALF was as high as 96%. The fibrinolytic activity of rALF revealed by the modified fibrin plate method indicated that the protein was efficiently secreted and functionally expressed, and thrombolysis of rALF was demonstrated to be dose-dependent and time-relative. The improved expression system will facilitate further studies and industrial production of ALF.
Shi
Jing
J
Northwest A&F University, Key Laboratory for Molecular Biology of Agriculture in Shaanxi Province, Yangling, Shaanxi Province, PR China.
Zhang
Shou-Tao
ST
Zhang
Xiao-Juan
XJ
Xu
Hong
H
Guo
Ai-Guang
AG
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis / Q000502:physiology; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C436755:alfimeprase