| Summary: | 碩士 === 國立交通大學 === 生物科技研究所 === 87 === Mammalian Secretory phospholipases A2(sPLA2,14KDa) are classified into two types : the pancreatic group I(PLA2-I) and the arthristic group II, on the basis of their primary structure.
Recent reports indicate that PLA2-I, in addition to its digestive function, has receptors-mediated effects. For instance, PLA2-I,via its receptor, can induce cell proliferation, airway and vascular smooth muscle contraction, chemokinesis and fertilization.
PLA2-I receptor is present in various kinds of organs, including mammalian stomach, pancreas, spleen and lung . These receptors with molecular masses of 180-200KDa, have been identified in rabbit, rat, mouse, human and bovine tissues.
To elucidate the distribution of PLA2-I receptor in different tissues and cells , especially in cancer cell ,we developed a simple non-radioisotope approach. A truncated Biotin carboxylase carrier protein (BCCPt , a.a.: 80-157) was used as a tag to label PLA2-I by fusing both porcine PLA2-I gene with truncated BCCP fragment cDNA.
To find out the smallest domain of BCCPt that can be still recognized and labeled by biotin ligase , we prepared various pPLA2-I-BCCPt fusion constructs containing different length of BCCP domain, including BCCPt(80-136), BCCPt(80-144), BCCPt(80-151) and BCCPt(80-156).
These expression constructs were over-expressed in E.Coli strain BL21(DE3).
The result showed that PLA2-I-BCCPt could be over expressed in BL21(DE3), although in a form of inclusion body. The biotinylation of all four fusion proteins were observed as detected by Streptavidin-conjugated HRP followed by TMS colorimeteric development.
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