Sreenivas Suma, Krishnaiah Sateesh M, Shyam Mohan Anil H, Mallikarjun Niveditha, Govindappa Nagaraja, Chatterjee Amarnath, Sastry Kedarnath N
Biocon Research Limited, Plot No. 2&3, Phase IV, Bommasandra-Jigani Link Road, Bangalore, 560099, Karnataka, India.
Molecular Diagnostics Laboratory, Dept. of Microbiology & Biotechnology, Bangalore University, JnanaBharathi Campus, Bangalore, 560 056, Karnataka, India.
Protein Expr Purif. 2016 Feb;118:1-9. doi: 10.1016/j.pep.2015.10.002. Epub 2015 Oct 22.
Insulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the C-terminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the C-terminal clipped species reduced by ∼80%. This modification further improved the process by reducing the levels of product related impurities.
甘精胰岛素是一种用于糖尿病治疗的长效胰岛素类似物。它通过重组DNA技术在不同宿主中生产，即大肠杆菌和巴斯德毕赤酵母。在我们之前的研究中，我们描述了通过过量表达Kex2蛋白酶将完全折叠的双链甘精胰岛素分泌到培养基中。Kex2蛋白酶水平的提高负责宿主内甘精胰岛素前体的加工。除了双链甘精胰岛素产物外，我们还观察到一小部分精氨酸被剪切的物种。这可能是由于B链C末端存在的精氨酸在Kex2切割后暴露而被剪切。已知羧肽酶前体Kex1负责剪切蛋白质或肽的C末端赖氨酸或精氨酸。为了解决这个问题，我们利用靶向基因缺失的Cre/loxP机制在宿主中创建了Kex1基因敲除。当在巴斯德毕赤酵母GS115的Kex1基因敲除宿主中表达双链甘精胰岛素时，C末端被剪切的物种减少了约80%。这种修饰通过降低产物相关杂质的水平进一步改进了工艺。
