Cloning, expression, and purification ofChlamydomonas reinhardtiiCC-503 sedoheptulose 1,7-bisphosphatase inEscherichia 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)VGN 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.

Heterologous mannitol-1-phosphate dehydrogenase gene over-expression in Parachlorella kessleri for enhanced microalgal biomass productivity

“…The samples were denatured at 95 °C for 5 min. The extracted proteins were separated on 12% acrylamide gel by SDS-PAGE and the proteins were stained by standard silver staining protocol [ 23 ].…”

An efficient algae cell wall disruption methodology for recovery of intact chloroplasts from microalgae

“…For experiment, the algal cultures were cultivated in 2,000 ml of polythene reactor in a specially designed environmental chamber (EC) (Fig. 1) which mimics the natural growth environment [8] with a light irradiance ≥1,200 µmol photons m −2 s −1 . The cells were grown until exponential growth phase (~10 10 cells ml −1 ), after which the cells were harvested by the centrifugation for 5 minutes at 10,000 g. The cells were washed three times with 0.1 M potassium phosphate buffer and pH 7.4 and resuspended in icecold grinding buffer [9] before lytic treatment.…”

A Review on Molecular Tools of Microalgal Genetic Transformation and their Application for Overexpression of Different Genes

“…Besides medium engineering and bioreactor designing, genetic engineering is an emerging alternative strategy of combating the problem of low biomass productivity in this field. Genetic engineering of microalgae is a fairly new approach and has been explored in a limited capacity to produce fatty acids enzymes, and vaccines in a limited number of microalgae mainly Chlamydomonas reinhardtii, Dunaliella salina, and Phaeodactylum tricornutum (Rasala et al, 2012;Geng et al, 2003;Hamilton et al, 2014;Vira et al, 2016). The advantages of using these photosynthetic cell factories include biosafety and low cost of substrates in the form of carbon dioxide, light and water…”