Viaplana E, Villaverde A
Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, Spain.
Biotechnol Prog. 1996 Sep-Oct;12(5):723-7. doi: 10.1021/bp9600593.
Three DNA segments encoding fragments of the p60 capsid protein of rabbit haemorrhagic disease virus (RHDV) have been cloned and expressed in Escherichia coli. The cDNAs were placed under the control of the T7 promoter in a pET-derived expression vector designed to produce C-terminal histidine tail fusions. By using different cloning strategies, cell toxicity exhibited by some of the expressed products was dramatically affected, coincident with minor modifications in the carboxy terminus sequences of the recombinant proteins. In particular, the presence of a nonhydrophobic peptide encoded by polylinker sequences promotes cell death and a reduced yield after induction of gene expression, whereas a histidine tail has no detectable effect. These data point out the critical role that needless peptide tails, accidentally introduced into recombinant proteins by nonrelevant DNA stretches, can have on protein expression and final yield of a production process.
编码兔出血性疾病病毒（RHDV）p60衣壳蛋白片段的三个DNA片段已在大肠杆菌中克隆并表达。这些cDNA被置于pET衍生表达载体中T7启动子的控制下，该载体设计用于产生C端组氨酸尾融合蛋白。通过使用不同的克隆策略，一些表达产物表现出的细胞毒性受到显著影响，这与重组蛋白羧基末端序列的微小修饰一致。特别是，多克隆位点序列编码的非疏水肽的存在会促进细胞死亡，并在基因表达诱导后降低产量，而组氨酸尾则没有可检测到的影响。这些数据指出了无关DNA片段意外引入重组蛋白中的不必要肽尾对蛋白质表达和生产过程最终产量可能产生的关键作用。
