Development of a Mass Production Method of Platelet Activating Factor Acetylhydrolase

Yong-Hwa Jong; Hyeun-Wook Chang; Tae-Yoon Lee

doi:10.4167/jbv.2006.36.4.229

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Jong, Chang, and Lee: Development of a Mass Production Method of Platelet Activating Factor Acetylhydrolase

Original Article

J Bacteriol Virol 2006;36(4):229-235.

Published online: 18 February 2006

DOI: https://doi.org/10.4167/jbv.2006.36.4.229

Development of a Mass Production Method of Platelet Activating Factor Acetylhydrolase

Yong-Hwa Jong, Hyeun-Wook Chang¹, Tae-Yoon Lee

Department of Microbiology, College of Medicine, Yeungnam University, 317-1 Daemyung-5-dong, Nam-gu, Daegu 705-035, Korea

¹College of Pharmacy, Yeungnam University, Gyongsan 712-749, Korea

Received 10 November 2006 Accepted 13 December 2006

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid mediator in a variety of physiological events. PAF is also involved in various pathological events including allergy and inflammation. PAF-acetylhydrolase (PAF-AH) hydrolyzes PAF to produce inactive lyso-PAF. Thus, overproduction of PAF-AF will be useful for the therapeutic valuation of the enzyme. In this study, we established an overproduction method of bovine PAF-AH in Escherichia coli system. We used bovine mammary gland for cDNA cloning. The cDNA had two mismatches of amino acid sequences (Thr-247 to Met and Ile-431 to Thr) compared with the previously reported PAF-AH cDNA (bovine spleen, NM_174578). The recombinant PAF-AH of 43 kDa in molecular size reacted with human PAF-AH polyclonal antibody and showed a strong PAF-AH enzyme activity in an in vitro assay system. The recombinant PAF-AH produced by this study can be applied for various experiments including in vivo models to test its protective activity against PAF-related diseases.

Keywords: PAF-AH, Anti-inflammatory protein, Mass production

References

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Figure 1.

PAF-AH activity from the lysates of E. coli BL21 cells. The PAF-AH activity in culture medium, lysates of BL21 cells, lysates of BL21 cells transformed with bovine PAF-AH gene on an expression vector pET30a were analyzed as described in Materials and Methods.

Figure 2.

SDS-PAGE profiles of cell lysates of E. coli BL21 transformed with pET30a (1), BL21 transformed with bovine PAF-AH-pET30a (2), and BL21 transformed with bovine PAF-AH-pET30a induced by IPTG (3). Induced expression of PAF-AH protein (43 kDa) is indicated by an arrow. M, protein size marker (104, 80, 46.9, 33.5, and 28.3 kDa from top to bottom).

Figure 3.

Western blot of recombinant PAF-AH expressed in cell lysates of E. coli BL21 transformed with bovine PAF-AH gene cloned on pET30a. PAF-AH protein (43 kDa) was detected using anti-human PAF-AH polyclonal antibody. M, prestained protein size marker (104, 80, 46.9, 33.5, and 28.3 kDa from top to bottom).

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