J Bacteriol Virol.  2006 Dec;36(4):229-235. 10.4167/jbv.2006.36.4.229.

Development of a Mass Production Method of Platelet Activating Factor Acetylhydrolase

• Jong YH
• Chang HW
• Lee TY

• KMID: 2055031
• DOI: http://doi.org/10.4167/jbv.2006.36.4.229

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.

Keyword

PAF-AH; Anti-inflammatory protein; Mass production

MeSH Terms

Amino Acid Sequence
Blood Platelets*
Clone Cells
Cloning, Organism
DNA, Complementary
Escherichia coli
Humans
Hypersensitivity
Inflammation
Mammary Glands, Human
Platelet Activating Factor*
Spleen
DNA, Complementary
Platelet Activating Factor

Figure

• 

  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).

Reference

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