[Cloning and prokaryotic expression of major ampullate spidroin gene of spider].
RT-PCR was conducted with one degenerate primer designed according to repetitive regions' amino acid sequence of major ampullate spidroin (MaSp) in spiders and adaptor primer in the SMART cDNA Library Construction Kit. By cloning and sequencing of amplified products, one cDNA clone (GenBank Accession No. AY365017) of Argiope amoena MaSp gene was obtained. The deduced amino acid sequence can be distinctly divided into two regions: (1) Repetitive region that consists of an alternating alanine-rich and glycine-rich domain in which many prolines are present; and (2) C-terminal non-repetitive region. The region coding for 272 amino acids of MaSp gene was subcloned into prokaryotic expression vector pET28b(+) and an about 26kD recombinant protein was expressed at high levels in Escherichia coli BL21 (DE3) after induction of IPTG. After being purified with metal-affinity chromatography on Ni(2+) -IDA-Sepharose columns as well as gel filtration chromatography, the recombinant protein was confirmed to be predicted MaSp by means of amino acid composition analysis and N-terminal amino acid sequence analysis. The solubility behavior of recombinant MaSp with C-terminal non-repetitive region in the present study is similar to that of recombinant dragline silk proteins without C-terminal non-repetitive region expressed by bacteria and yeast in the other studies. The result shows that absence or presence of C-terminal non-repetitive region is not a crucial factor affecting the solubility of the recombinant MaSp.
Pan
Hong-Chun
HC
Laboratory of Molecular Evolution & Biodiversity, College of Life Sciences, Anhui Normal University, Wuhu 241000, China. panhongchun@126.com
Song
Da-Xiang
DX
Zhou
Kai-Ya
KY
Zhu
Guo-Ping
GP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005349:Fibroins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013112:Spiders / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005349:Fibroins