title: In vitro refolding of triosephosphate isomerase from L. donovani.
abstract: The triosephosphate isomerase of Leishmania donovani (LdTIM) was expressed at high level in Escherichia coli. The TIM gene was cloned in expression vector pET-23(a) with C-terminal 6× His tag fused in frame, and expressed as a 27.6-kDa protein in E. coli as inclusion bodies. The recombinant LdTIM from E. coli lysate was solubilized in 6 M guanidine hydrochloride and purified by Ni-NTA chromatography. In the present study, the effect of bovine serum albumin on the reactivation of TIM was investigated. Furthermore, 8-anilino-1-naphthalene sulfonic acid was used to detect the structural changes induced by bovine serum albumin (BSA). Here, we conclude that BSA assists in the refolding and regain of LdTIM enzyme activity by providing framework for structure formation. This study indicates that numerous protein-protein contacts are constantly occurring inside the cell that leads to the formation of native protein.
authors.affiliation: Division of Biochemistry, Central Drug Research Institute, Lucknow 226001, UP, India.
authors.forename: Kishore
authors.initials: K
authors.lastname: Kumar
authors.forename: Prachi
authors.initials: P
authors.lastname: Bhargava
authors.forename: Uma
authors.initials: U
authors.lastname: Roy
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000032:analysis; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D019791:Guanidine / Q000737:chemistry; D002479:Inclusion Bodies / Q000201:enzymology* / Q000235:genetics; D007893:Leishmania donovani / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017510:Protein Folding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D014305:Triose-Phosphate Isomerase; D019791:Guanidine