کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2021997 | 1069273 | 2007 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Expression and characterization of a recombinant unique acid phosphatase from kidney bean hypocotyl exhibiting chloroperoxidase activity in the yeast pichia pastoris Expression and characterization of a recombinant unique acid phosphatase from kidney bean hypocotyl exhibiting chloroperoxidase activity in the yeast pichia pastoris](/preview/png/2021997.png)
We previously purified and characterized a novel acid phosphatase (KhACP) from kidney bean hypocotyls that exhibited vanadate-dependent chloroperoxidase (V-CPO) activity. In the present study, a functional recombinant KhACP (rKhACP) was successfully produced at a high expression level by the methylotrophic yeast Pichia pastoris. The KhACP cDNA excising signal peptide sequence was subcloned into the pPICZαA vector and then integrated into the genome of P. pastoris strain X-33 under control of the alcohol oxidase 1 promoter. The rKhACP protein, with a molecular mass of 60 kDa on SDS–PAGE, was secreted into the culture medium as a C-terminal His-tagged fusion protein. Purification was facile using only nickel affinity chromatography. The apparent molecular mass of the purified rKhACP was estimated to be around 110 kDa by analytical gel filtration. PAGE analysis showed that rKhACP was a glycosylated dimeric enzyme, consisting of two 60-kDa subunits linked non-covalently, which was similar to the dominant form of the natural enzyme isolated from plant material. Furthermore, the rKhACP exhibited V-CPO activity when ortho-vanadate (VO43-) was added to the apo enzyme, and it showed broad substrate specificity and kinetic parameters comparable to the natural enzyme. This expression system produces sufficient protein to allow us to attempt to determine the three-dimensional crystal structure, which will shed light on its unique mechanism of converting KhACP to vanadate-dependent chloroperoxidase.
Journal: Protein Expression and Purification - Volume 53, Issue 1, May 2007, Pages 31–39