Evaluation of the myristoylation on conferring proteins with membrane-binding property

• Main Authors: Chih-Heng Huang, 黃致衡
• Other Authors: 呂維茗
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/81673985020302840339

碩士 === 國立中興大學 === 生物科技學研究所 === 101 === Myristoylation is an irreversible attachment of a myristic acid (C14:0) onto the N-terminal glycine of a protein via an amide bond. This modification changes the inherent hydrophobicity of a protein, often renders its targeting to the membrane compartments and may modify its protein activity, stability, and protein–protein interactions. Using transiently bombarded lily pollen, a calcium-regulated protein kinase OsCPK26 from rice was bound on the plasma membrane predominantly. A G2A mutation fully disrupted such binding, suggest a necessity of myristoylation. Serial C-terminal deletions revealed that merely the N-terminal seven amino acids (26(1-7)) was enough for efficient membrane-targeting of the fused ECFP moiety. We aim to investigate is it a consistent phenomenon applicable to other plant or even prokaryotic cells? Can the myristoylation signal peptide be an effective fusion tag for protein expression and/or purification? Plasmolysis applied on the bombarded onion epidermis rendered recognizable signals of 26(1-7)/mGFP5, but barely the non-fused mOrange2, to the Hechtian strands constituted by the plasma membrane. Moreover, 26(1-7)/mGFP5 was co-localized with the plasma membrane marker FM4-64 in the tobacco protoplast. Both evidences suggest a membrane-binding status of 26(1-7)/mGFP5, although not to an extensive level. Searching of other myristoylation signals revealed that only HV1A2(1-7), but not FLV76(1-7) or YEAST(1-7), conferred mGFP5 a membrane-binding property in the plant cells. In the E. coli cells, co-expression of the yeast N-myristoyltransferase (NMT) did render ~50% of the OsCPK26, OsCPK26 variable domain, OsCPK26(1-7)/mGFP5, and HV1A2(1-7)/mGFP5 proteins distributed into the membrane fraction under ultracentrifugation. Possible improvements of the membrane-targeting by myristoylation are discussed. The E. coli harboring pACYC177-ET3d-yNMT produced a noticeable banding on SDS-PAGE, confirmed to be NMT by mass spectrum analysis. It is interesting to note that the overexpression of NMT was observed only when the host BL21(pLysS) was co-transformed with a second plasmid, but not the pACYC177-ET3d-yNMT alone. Looking for clues for the NMT overexpression phenomenon did rule out the involvement of the NMT substrate protein, antibiotics selection, IPTG induction, and promoter control, etc. Surprisingly, BL21 strain overexpressed the NMT in absence of a second plasmid. We suspect that pLys plasmid may compete, or recombine, with the pACYC177-ET3d-yNMT as both plasmids share an identical 835-bp fragment including the replication origin. Restriction mappings on the co-transformed plasmids revealed both missing and extra bandings, supporting the above possibilities.