Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris

• Published in: Protein expression and purification Vol. 118; pp. 1 - 9
• Main Authors: Sreenivas, Suma, Krishnaiah, Sateesh M., Shyam Mohan, Anil H., Mallikarjun, Niveditha, Govindappa, Nagaraja, Chatterjee, Amarnath, Sastry, Kedarnath N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2016
• Subjects: Carboxypeptidases - genetics; Carboxypeptidases - metabolism; Cre/lox mechanism; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene inactivation; Gene Silencing; Humans; Insulin Glargine - metabolism; Marker rescue; Pichia - enzymology; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Protein Transport; Proteolysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Two-chain glargine; Gene inactivation; Cre/lox mechanism; Two-chain glargine; Pichia pastoris; Marker rescue
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.10.002

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. •Kex1 gene inactivation to reduce proteolysis of two-chain glargine in Pichia pastoris.•Process improvement by reducing product related impurities by gene inactivation.•Cre/lox mechanism for targeted gene inactivation and marker recycling in Pichia.