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Expression Of Lumbrokinase Gene In Pichia Pastoris

Posted on:2008-01-06Degree:MasterType:Thesis
Country:ChinaCandidate:C Y ZhangFull Text:PDF
GTID:2120360245493414Subject:Biochemical Engineering
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Lumbrokinase was extracted and purified from the earthworm, as a group of potential fibrinolytic enzymes, it was being extensively investigated. Therefore, it has quite potential property to facilitate it being developed into anti-thrombotic drugs. Lumbrokinase has already come into market in South Korea and China.Pichia pastoris has been developed into a highly successful system for expression of heterologous proteins. It was able to perform higher eukaryotic protein modifications, such as glycosylation, disulphide bond formation and proteolytic processing. Most researchers use GS115 as expression host and pPIC9K as expression vector. In this paper, a system for expression of recombinant lumbrokinase was constructed in the yeast Pichia pastoris GS115.The gene encoding the native form of Lumbrokinases was obtained by PCR amplification and was sub-cloned into pPIC9K, a yeast expression and secretion vector. Then the constructed expression vector was linearized by BglⅡand was electroporated into host strain GS115. Transformer was obtained after screened by histidine nutrition deficient plate and antibiotic G418. PCR result showed the interested gene has been inserted into the host strain. Nouthern Dot Blotting and SDS–PAGE analyses showed that LK gene has been cloned into the genome of Pichia pastoris and has been transcripted and expressioned successfully. The transformer we obtained was identified as methanol utilization slow phenotype (Muts) by methanol nutrition limited plate. Being induced by methanol the culture medium of different fermentation stage was able to dissolve artificial fibrin plates.This work may provide a significant foundation for the bioengineering production of lumbrokinase.
Keywords/Search Tags:Lumbrokinase, Pichia pastoris, expression, anti-thrombotic
PDF Full Text Request
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