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Cloning, expression, and purification of lipoprotein-associated phospholipase A2 in Pichia pastoris

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Abstract

Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been shown to play a crucial role in atherosclerosis, and has been proposed as a promising target for drug discovery. Here, we cloned the Lp-PLA 2 gene from differentiated THP-1 cells, and inserted a carboxy-terminal His6-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA2 fusion protein was successfully expressed in Pichia pastoris expression system and could be rapidly purified to apparent homogeneity using a single-step purification method. The activity of our recombinant Lp-PLA2 was strong when [3H] PAF was used as a substrate, and the Lp-PLA2 inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA2 (IC50=15.93±1 μM). This novel recombinant Lp-PLA2 could prove useful as a screening model for Lp-PLA2 inhibitors, and may facilitate further investigation of this protein in atherosclerosis.

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Authors and Affiliations

  1. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, 201203, Shanghai, China

    Fujun Zhang & Yiping Wang

  2. Graduate School, Chinese Academy of Sciences, 201203, Shanghai, China

    Fujun Zhang

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  1. Fujun Zhang
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  2. Yiping Wang
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Correspondence to Yiping Wang.

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Zhang, F., Wang, Y. Cloning, expression, and purification of lipoprotein-associated phospholipase A2 in Pichia pastoris . Mol Biotechnol 33, 29–36 (2006). https://doi.org/10.1385/MB:33:1:29

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