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| Exogenous Alanine Enhances the Expression of Spidroin2 cDNA of Spider Dragline Silk Protein in Escherichia coli |
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Abstract Abstract Expression in vitro of the natural genes of spider silk protein is limited by many factors. Based on the cloning of Spidroin2 cDNA of Nephila clavipes (Genbank Accession No. AF441245), we constructed a prokaryotic expression vector pET-28b(+)-Sp by double digestion. The recombinant plasmids pET-28b(+)-Sp were then transferred into the competent expression host strain BL21(DE3) E. coli. Expression of the native gene Spidroin2 cDNA was induced by the addition of different concentrations of IPTG, followed by extension of induction time, culture temperature and addition of exogenous alanine to increase the expression of interest gene. The recombinant protein was identified by SDS-PAGE and Western blot in which the polyclonal antiserum of dragline silk protein was used as the first antibody. Sequencing result of recombinant expression plasmid showed that Spidroin2 cDNA was inserted into the prokaryotic expression vector pET-28b(+) with a correct read frame. An interest protein with a molecular weight of 31 kDa was observed on SDS-PAGE gel in the condition of alanine addition, which can be specifically immunoblotted with a polyclonal antibody targeted at spider silk protein. We concluded that the Spidroin2 cDNA of Nephila clavipes can be expressed in prokaryotic expression system, and addition of exogenous alanine plays important role in the promotion of expression of the naive gene of spider silk protein.
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Received: 14 July 2006
Published: 10 May 2010
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