Mutagenesis And Kinetic Analysis of Low Molecular Weight Phosphotyrosyl Protein Phosphatase

• Main Authors: Xiang-Kai Lin, 林翔凱
• Other Authors: Yu-Yuan Wo
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/78343594454554124913

碩士 === 國立嘉義大學 === 生化科技學系研究所 === 98 === Due to alternative splicing of the primary RNA transcript encoded by the ACP1 locus , there are two types of LMW-PTP, S form and F form. In human They are named HPTP-F and HPTP-S. They are different in residue 40~73. Although the cellular activities of LMW-PTPs were yet determined, recent studies have demonstrated that their activities are related to immune response, cardiovascular disorders, infections, and cancer. However the correlation between structure and topology of LMW-PTP still remains unclear. In this study, we have used PCR-mediated gene construction for the construction of LMW-PTP expression plasmid DNA. To correlate the structure and enzymatic activity of LMW-PTPs, two methods, random mutagenesis by PCR and point mutation analysis, were used to elucidate the critical residues in LMW-PTPs. Some mutants exhibited either great decrease or increase in enzymatic activity, as compared with wild type construct, were further purified and subject to Michaelis-Menten activity assay. By this procedure, the structural significance of some residues in LMW-PTPs is thus established. As that is predicted earlier, we found the residue Asn134 actually played an important role in structure and enzymatic activity. Mutants N134D and N134S exhibited a significant influence on the activities. However, the mutation effects of N to S or D are different in relative activity. More interestingly, their effects on LMW-PTP S and LMW-PTP F are different as well. In the future, it is suggested that the obtained mutant, which exhibited higher activity than the wild type LMW-PTP, might provide a valuable tool in therapeutic or academic applications.