[Construction, expression and purification of repeat fragment polymer of oInhibin alpha-subunit N terminal 1-33 fragment].
A cDNA sequence coding for ovine inhibin N terminal 1-33 AA residue fragment (INH) was inserted between BamHI\SacI sites in plasmid pRSET-A to generate plasmid pR-INH. By utilizing a pair of isocaudamer BamHI and Bgl II sites and another downstream Hind III site, following simple double digestions and combination ligation of the resultant products, 2 to 6-repeat INH genes were constructed respectively. Each plamids containing 3 to 6 repeated INH fragment genes, pR-3INH, pR-4INH, pR-5INH and pR-6INH, directed expression of the target proteins in E. coli. BL21 (DE3) under induction of ITPG, which respectively accounted for 6%, 6%, 7% and 8% of the total bacterial protein. The expressed target proteins were all in the form of inclusion bodies. The above results implied that utilization of isocaudamer restriction disgetion sites in expression plasmid is capable of rapidly and correctly constructing repeat fragment polymer of short peptides, which may become a new method in construction of high immunogenic recombinant vaccines of short peptides.
Huang
Yun-Mao
YM
College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
Shi
Zhen-Dan
ZD
Yu
Ying-Chun
YC
Shao
Xi-Bing
XB
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007265:Inhibins / Q000096:biosynthesis* / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011108:Polymers / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012756:Sheep
D010446:Peptide Fragments; D011108:Polymers; D011993:Recombinant Fusion Proteins; C094377:inhibin-alpha subunit; D007265:Inhibins