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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >A BIFUNCTIONAL FUSION PROTEIN CONTAINING THE MALTOSE-BINDING POLYPEPTIDE AND THE CATALYTIC CHAIN OF ASPARTATE TRANSCARBAMOYLASE - ASSEMBLY, OLIGOMERS, AND DOMAINS
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A BIFUNCTIONAL FUSION PROTEIN CONTAINING THE MALTOSE-BINDING POLYPEPTIDE AND THE CATALYTIC CHAIN OF ASPARTATE TRANSCARBAMOYLASE - ASSEMBLY, OLIGOMERS, AND DOMAINS

机译：包含麦芽糖结合多肽和天冬氨酸转氨羧酶的催化链的功能性融合蛋白-组装，寡聚体和域

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The in vivo synthesis of many target proteins or polypeptides has been enhanced dramatically and their purification facilitated through the use of gene fusion techniques which lead to the expression of fusion proteins, This approach was used to characterize the product formed in Escherichia coli encoded by a DNA construct comprising malE, the gene encoding maltose binding protein, linked to a small 30 nucleotide region which, in rum, was linked to pyrB, the gene encoding the catalytic (c) chains of aspartate transcarbamoylase (ATCase). The resulting fusion protein, MBP-C, was produced in excellent yield and readily purified in two steps because of its binding to an amylose column and displacement by maltose. The complex was studied by both sedimentation velocity and sedimentation equilibrium and shown to be a trimer of c chains with one MBP linked covalently to each chain, Treatment of the fusion protein with factor Xa cleaved each chain at the tetrapeptide encoded by the linker region yielding purified MBP with a minor modification at the C-terminus and the catalytic (C) trimer of ATCase, The MBP-C complex was fully active as an enzyme and could be reversibly denatured in 6 M urea. Scanning calorimetry studies on the fusion protein demonstrated that the MBP domain melted at the same temperature as did the purified protein. Similarly, the T-m for the C trimer in the complex was identical to the value for C trimer isolated from ATCase. Moreover, the thermal stability of the C trimer in the MBP-C complex was greatly enhanced by the addition of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), just as observed with purified C trimer, Analogous denaturation experiments with varying concentrations of guanidine-HCl indicated that the fusion protein was denatured at much lower concentration of denaturant than observed for C trimer. These experiments demonstrate that the linker between the two structural genes encodes a polypeptide of sufficient length to permit independent folding and assembly of each protein and permit the subsequent specific cleavage at the factor Xa recognition site, thereby yielding both active proteins. [References: 29]
机译：通过使用导致融合蛋白表达的基因融合技术，大大提高了许多靶蛋白或多肽的体内合成，并促进了其纯化。该方法用于鉴定在DNA中编码的大肠杆菌中形成的产物。包含malE的基因构建体，该基因编码麦芽糖结合蛋白的基因，连接到一个小的30个核苷酸区域，在朗姆酒中连接到pyrB，该基因编码天冬氨酸转氨甲酰酶（ATCase）的催化（c）链。生成的融合蛋白MBP-C以极高的产率产生，并且由于其与直链淀粉柱的结合以及麦芽糖的置换，因此易于分两步纯化。通过沉降速度和沉降平衡对复合物进行了研究，结果显示该复合物是c链的三聚体，每个MBP共价连接一个MBP。用Xa因子处理融合蛋白可在连接子区域编码的四肽处裂解每条链，从而得到纯化的MBP-C复合物在酶的C末端和ATCase的催化（C）三聚体处有微小修饰，可作为一种酶完全发挥活性，并在6 M尿素中可逆地变性。对融合蛋白的扫描量热法研究表明，MBP结构域在与纯化蛋白相同的温度下融化。类似地，复合物中C三聚体的T-m与从ATCase分离的C三聚体的值相同。此外，通过添加双底物配体N-（膦酰基乙酰基）-L-天门冬氨酸（PALA），MBP-C络合物中C三聚体的热稳定性大大提高，与纯化的C三聚体类似，通过变性实验浓度不同的胍盐酸盐溶液表明融合蛋白在变性剂浓度比在C三聚体中观察到的低得多。这些实验表明，两个结构基因之间的接头编码了足够长的多肽，以允许每种蛋白质独立折叠和组装，并允许随后在因子Xa识别位点进行特异性切割，从而产生两种活性蛋白质。 [参考：29]

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