莱茵衣藻CC-503 sedoheptulose 1,7-双磷酸酶在大肠杆菌中的克隆、表达和纯化

Chaitali Vira, G. Prakash, J. Rathod, A. Lali
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引用次数: 3

摘要

Sedoheptulose 1,7-bisphosphatase (SBPase)是一种核编码的叶绿体酶,是碳固定循环(Calvin cycle)中重要的限速酶。SBPase仅存在于光合生物中,参与核酮糖-1,5-二磷酸的再生。已经研究了几种来源的sbpase的诱导和调控。然而,被广泛研究的模型微藻莱茵衣藻CC-503中的SBPase尚未被分离出来并在功能上得到证实。本研究利用锚定oligo(dT)24VGN引物从C. reinhardtii CC-503中分离出SBPase全长cDNA进行反转录。将SBPase cDNA克隆到大肠杆菌BL21 (DE3)菌株的pET28a表达载体中,用于生产6X his标记蛋白。虽然最初获得的大部分酶是不溶性蛋白质聚集体,但通过优化蛋白质诱导,提高了蛋白质的增溶性,这与生长温度和异丙基β- d -1-硫代半乳糖苷浓度有关。用镍-硝基三乙酸树脂在无磷酸盐缓冲液中固定化金属亲和层析纯化诱导蛋白,从而精确测量SBPase活性。本研究首次在大肠杆菌中成功克隆了C. reinhardtii CC-503 SBPase,并表达了具有功能活性的SBPase。
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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.
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