Construction of synthetic genes for plasmodium falciparum dihydrofolate reductase-thymidylate synthase and Pneumocystic carinii dihydrofolate reductase
1
Issued Date
2023
Copyright Date
1993
Language
eng
File Type
application/pdf
No. of Pages/File Size
xxv, 375 leaves : ill. (some col.)
Access Rights
restricted access
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (Ph.D. (Biochemistry))--Mahidol University, 1993
Suggested Citation
Phisit Prapunwattana Construction of synthetic genes for plasmodium falciparum dihydrofolate reductase-thymidylate synthase and Pneumocystic carinii dihydrofolate reductase. Thesis (Ph.D. (Biochemistry))--Mahidol University, 1993. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/89656
Title
Construction of synthetic genes for plasmodium falciparum dihydrofolate reductase-thymidylate synthase and Pneumocystic carinii dihydrofolate reductase
Alternative Title(s)
สายยีนสังเคราะห์สำหรับเอนไซม์ไดไฮโดรโฟเลทรีดักเตส-ไธมิไดเลทซินเทสของเชื้อพลาสโมเดียมฟาลซิปารั่มและเอนไซม์ไดไฮโดรโฟเลทรีดักเตสของเชื้อนิวโมซิสติสคารนิอี
Author(s)
Advisor(s)
Abstract
Based on the known protein sequences, two synthetic genes were designed and constructed. One encoded Pneumocystis carinii dihydrofolate reductase (PcDHFR) and another encoded the bifunctional Plasmodium falciparum dihydrofolate reductasethymidylate synthase (PfDHFR-TS). The synthetic P. falciparum DHFR-TS was first constructed as 3 separate domains: DHFR (PfDHFR), junction and TS (PfTS) domains and all domains were joined together to create the synthetic entire DHFR-TS sequence. DNA sequences of all synthetic genes/domains were designed based on biased codon usage of Escherichia coli and to contain unnatural restriction sites interspersed evenly through out their coding regions. The synthetic PcDHFR, PfDHFR, PfTS and PfDHFR-TS genes were also designed so as to contain their own translation unit and also contain transcription terminator. The synthetic PcDHFR was mutated to three single mutants and one triple mutant (combining all three mutations). The residues Ile-33, Lys-37 and Phe-69 were changed to their counterpart residues in human DHFR, Phe, Gln and Asn, respectively. These three residues were earlier proposed to be responsible for the difference in sensitivity to trimethoprim and its analogues of Pneumocystis and human DHFRs. The wild-type PcDHFR and mutants were expressed and purified to homogeneity. Steady state kinetic parameters of all enzymes were determined, and each was examined for inhibition by several related antifolates. The data indicatsed that these three amino acids do contribute to the species selectivity as predicted. The synthetic PfDHFR domain was expressed as active monofunctional DHFR enzyme in E. coli to a level that could overcome the inhibitory effect of trimethoprim present in the medium. The synthetic PfTS domain was expressed as inclusion bodies under T7 promoter control. The TS activity could not be recovered by refolding approach. The complete synthetic PfDHFR-TS gene could be expressed as the active bifunctional enzyme, and the expression level was higher than previously reported for the cloned natural gene. The enzyme was partially purified and kinetic parameter was determined, including effect of salts and urea. The enzyme properties observed were comparable to the one previously obtained from the cloned natural gene.
Degree Name
Doctor of Philosophy
Degree Level
Doctoral Degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University