title: Cloning, expression, and purification of Chlamydomonas reinhardtii CC-503 sedoheptulose 1,7-bisphosphatase in Escherichia coli.
abstract: Sedoheptulose 1,7-bisphosphatase (SBPase), a nuclear-encoded chloroplastic enzyme, is an important rate-limiting enzyme of the carbon fixation cycle (Calvin cycle). SBPase is unique to only photosynthetic organisms and is involved in the regeneration of ribulose-1,5-bisphosphate. SBPases from several sources have been studied for their induction and regulation. However, SBPase from Chlamydomonas reinhardtii CC-503, the widely studied model microalga, has not been isolated and functionally confirmed to date. In this study, the full-length cDNA for SBPase was isolated from C. reinhardtii CC-503 using anchored oligo(dT)24VGN primer for reverse transcription. The SBPase cDNA was cloned into pET28a expression vector for the production of 6X His-tagged protein in Escherichia coli BL21 (DE3) strain. Although initially most of the enzyme was obtained as insoluble protein aggregates, solubilization of protein was improved by optimization of protein induction with respect to growth temperature and isopropyl β-D-1-thiogalactopyranoside concentrations. The induced protein was purified by immobilized metal affinity chromatography using nickel-nitrilotriacetic acid resin in a phosphate-free buffer leading to an accurate SBPase activity measurement. The present study demonstrates, for the first time, successful cloning of C. reinhardtii CC-503 SBPase in E. coli leading to the expression of a functionally active enzyme.
authors.affiliation: a DBT-ICT Centre for Energy Biosciences , Institute of Chemical Technology , Matunga , Mumbai , India.
authors.forename: Chaitali
authors.initials: C
authors.lastname: Vira
authors.affiliation: a DBT-ICT Centre for Energy Biosciences , Institute of Chemical Technology , Matunga , Mumbai , India.
authors.forename: Gunjan
authors.initials: G
authors.lastname: Prakash
authors.affiliation: a DBT-ICT Centre for Energy Biosciences , Institute of Chemical Technology , Matunga , Mumbai , India.
authors.forename: Jayant Pralhad
authors.initials: JP
authors.lastname: Rathod
authors.affiliation: a DBT-ICT Centre for Energy Biosciences , Institute of Chemical Technology , Matunga , Mumbai , India.|b Department of Chemical Engineering , Institute of Chemical Technology , Matunga , Mumbai , India.
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Lali
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D066329:Protein Aggregates; D012995:Solubility
chemical_list: D018076:DNA, Complementary; D066329:Protein Aggregates; D010744:Phosphoric Monoester Hydrolases; C022627:sedoheptulose-bisphosphatase
keywords: Chlamydomonas reinhardtii CC-503; IMAC; cDNA cloning; enzyme purification; oligo(dT)24VGN; sedoheptulose 1,7-bisphosphatase