[Study on construct and expression of synthetic genes encoding spider dragline silk in Escherichia coli].
Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity. Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed. DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG. The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L. The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90%. The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD. This value and amino acid analysis were consistent with those of theoretic calculation.
Li
Min
M
College of Biological Engineering, Fujian Normal University, Fuzhou 350007, China. MLI2@sina.com
Zhang
Wen-Xian
WX
Huang
Zhi-Hua
ZH
Huang
Jian-Kun
JK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005349:Fibroins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification
D000596:Amino Acids; D011506:Proteins; D011994:Recombinant Proteins; C076815:spidroin 2; D005349:Fibroins