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Molecular Cloning and Expression of Cardiotoxin Ⅲ from Naja naja atra in E.coli and Yeast Pichia pastoris / 中国生物工程杂志
China Biotechnology ; (12): 23-30, 2008.
Article in Chinese | WPRIM | ID: wpr-737100
ABSTRACT
Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60~62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.

Full text: Available Index: WPRIM (Western Pacific) Type of study: Practice guideline Language: Chinese Journal: China Biotechnology Year: 2008 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Type of study: Practice guideline Language: Chinese Journal: China Biotechnology Year: 2008 Type: Article