Investigation of halotag fusion protein function in E. coli and plasmodium
1
Issued Date
2014
Copyright Date
2014
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xv, 71 leaves : col. ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Biochemistry))--Mahidol University, 2014
Suggested Citation
Ahmed, Golam Rizvee, 1988- Investigation of halotag fusion protein function in E. coli and plasmodium . Thesis (M.Sc. (Biochemistry))--Mahidol University, 2014. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/95269
Title
Investigation of halotag fusion protein function in E. coli and plasmodium
Author(s)
Abstract
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.
Description
Biochemistry (Mahidol University 2014)
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University