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首页> 外文期刊>Protein Expression and Purification >Isolation of the gene and large-scale expression and purification of recombinant Erythrina cristagalli lectin
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Isolation of the gene and large-scale expression and purification of recombinant Erythrina cristagalli lectin

机译：重组赤藓促凝集素基因的分离及大规模表达与纯化

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Using polymerase chain reaction, the coding sequence for Erythrina cristagalli lectin (ECL) has been cloned and expressed in Escherichia coli. The amplified DNA sequence of ECL is highly homologous to that previously reported for Erythrina corallodendron lectin (ECorL), confirming the absence of introns in the ECL gene. The polypeptide sequences of ECL and ECorL have been compared and five amino acids have been identified that differentiate the two proteins. Recombinant E. cristagalli lectin (recECL) was expressed in E. coli from a genomic clone encoding the mature E. cristagalli lectin gene. Constitutive expression localised recombinant protein in inclusion bodies, which were solubilised, and recECL, subsequently refolded and purified by lactose affinity chromatography. Significant advantages were observed for purification from inclusion bodies rather than from a clone optimised to express soluble protein. A large-scale purification scheme has been developed that can prepare functional recECL from inclusion bodies with a yield of 870 mg/L culture. By the range of characterisation methods employed in this study, it has been demonstrated that recECL is functionally equivalent to native ECL obtained from the E. cristagalli plant. In addition, characterisation of the binding of radiolabelled recECL to cultured dorsal root ganglia demonstrated that recECL binds to a single pool of receptors. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 15]
机译：使用聚合酶链反应，已经克隆了大肠杆菌赤藓菌的编码序列（ECL）并在大肠杆菌中表达。 ECL的扩增DNA序列与先前报道的Erythrina珊瑚科植物凝集素（ECorL）高度同源，这证实了ECL基因中没有内含子。比较了ECL和ECorL的多肽序列，并鉴定了5种氨基酸来区分这两种蛋白质。重组大肠杆菌cristagalli lectin（recECL）由编码成熟大肠杆菌cristagalli lectin基因的基因组克隆在大肠杆菌中表达。组成型表达将重组蛋白定位在包涵体中，将其溶解，然后recECL，然后通过乳糖亲和层析重折叠并纯化。从包涵体而不是从优化表达可溶蛋白的克隆中纯化获得了显着优势。已经开发了一种大规模纯化方案，可以从包涵体制备功能性recECL，培养量为870 mg / L。通过本研究中使用的多种表征方法，已证明recECL在功能上等同于从E. cristagalli植物获得的天然ECL。此外，放射标记的recECL与培养的背根神经节的结合特征表明recECL与单个受体结合。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：15]

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