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KMID : 0545120000100050663
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 5 p.663 ~ p.669
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Construction of a New Gene-Fusion Expression Vector,pMONSTER
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Baek Chang-Ho
Wee Se-Chan
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Abstract
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The Fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion proteins with β-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur-β-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-β-D-galactoside plate and the resulting 133kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified FurHIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.
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