Thanat ChookajornOrnchuma ItsathitphaisarnShaw, Philip JamesAhmed, Golam Rizvee, 1988-2024-02-072024-02-07201420142014Thesis (M.Sc. (Biochemistry))--Mahidol University, 2014https://repository.li.mahidol.ac.th/handle/20.500.14594/95269Biochemistry (Mahidol University 2014)HaloTag (HT) is a modified bacterial haloalkane dehalogenase enzyme that has been developed as a multi-purpose tool for studying protein functions, including protein purification, intracellular protein localization, time-dependent labeling, and control of intracellular protein levels. Ligand-mediated control of intracellular protein is an attractive tool for drug target validation. The function of HT was initially tested in E. coli. EGFP-HT fusion protein was functional when expressed in E. coli, although the expression was markedly lower than expected. Another reporter protein, dihydrofolate reductase thymidylate synthase (DHFR-TS) was used as a fusion partner to HT and its function was tested in E. coli. It was observed that the fusion of HT to DHFR-TS protein lowered the expression and intracellular activity of DHFR-TS compared with a 6x His tagged DHFR-TS. The DHFRTS-HT fusions had correspondingly lower DHFR activity to complement a DHFR-TS E. coli mutant. The expression of the EGFP-HT fusion protein was also tested in the blood stages of the Plasmodium berghei rodent malaria parasite. Correct integrants were validated by PCR. EGFP-HT mRNA expression was detected by RT-PCR in transgenic EGFP-HT parasite lines. However, no EGFP-HT fusion protein was detected by Western blot. In conclusion, HT can be expressed as a fusion protein, but the low levels of protein activity suggest that HT interferes with fusion protein expression and/or stability in E. coli and Plasmodium sp.xv, 71 leaves : col. ill.application/pdfengผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้าProtein engineering.Polymerase chain reactionProteins -- Synthesis -- Laboratory manuals.Master ThesisMahidol University