Sharma Shamik, Zhang Aihua, Wang Haoyong, Harcum Sarah W, Chong Shaorong
New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USA.
Biotechnol Prog. 2003 May-Jun;19(3):1085-90. doi: 10.1021/bp034009r.
Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.
蛋白质剪接元件（内含肽）能够催化可控的肽键裂解反应，已被用于在亲和纯化过程中从亲和标签上分离重组蛋白。由于内含肽在回收过程中无需使用蛋白酶，内含肽介导的纯化系统有潜力显著降低重组蛋白工业生产的回收成本。到目前为止，内含肽系统仅在实验室规模下针对低细胞密度培养的细胞进行了重组蛋白表达和纯化的研究与应用。在本研究中，对重组大肠杆菌高细胞密度补料分批发酵中表达的内含肽融合蛋白的蛋白质剪接和体外裂解进行了研究。使用三种模型内含肽融合构建体来检测在高细胞密度条件下产生的融合蛋白的稳定性以及剪接/裂解活性。数据表明，含有野生型内含肽的内含肽融合蛋白在高细胞密度培养期间催化了高效的体内蛋白质剪接。此外，含有修饰内含肽的内含肽融合蛋白催化了高效的硫醇诱导的体外裂解反应。本研究结果证明了使用内含肽介导的蛋白质纯化系统进行重组蛋白工业规模生产的潜在可行性。
