Cloning And Expression Of Lumbrokinase Gene In Pichia Pastoris

In this research, cloning and expression of lumbrokinase gene in Pichia pastoris were studied. The main results were as follows.The total RNA was obtained from earthworm(Eisenia fetida). The whole gene of lumbrokinase F238 was amplified by RT-PCR.The whole gene of lumbrokinase F238 was inserted into pUCm-T vector to construct pUCm-T-F238. The product was sequenced. The GenBank accession No was DQ202401. The result showed that the fragment was 738bp, encoding 7 amino acid residues as signal peptide and 238 amino acid residues as mature peptide. There were two nucleotide mutations between F238 and Lumbricus rubellus F-III-2, which caused two amino acids mutations. The prediction of protein structure showed that lumbrokinase F238 had serine active center. It was a protease in trypsin family of serine protease superfamily.Lumbrokinase F238-m without the signal sequence was cloned by PCR using pUCm-T-F238 as template. Expression vector pPIC9-F238-m was constructed and transformed into Pichia pastoris GS115. Transformants were induced by methanol for the expression of lumbrokinase. The engineering strain of high expression was screened by SDS-PAGE method. The relative molecular weight was about 2.8×10~4.The engineering strain had obvious fibrinolytic activity with fibrin plate method. The optimum fermentation conditions were confirmed as follows: seed age 20h, initial pH6.5, temperature 29℃, 20mL BMMY medium in 250mL flask, methanol concentration 0.25 %. The fibrinolytic activity was up to 189U/mL after 84 hours.