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

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 â¼80. This modification further improved the process by reducing the levels of product related impurities. © 2015 Elsevier Inc. Elsevier Inc. All rights reserved

Failure Detectors Encapsulate Fairness

Failure detectors have long been viewed as abstractions for the synchronism present in distributed system models. However, investigations into the exact amount of synchronism encapsulated by a given failure detector have met with limited success. The reason for this is that traditionally, models of partial synchrony are specified with respect to real time, but failure detectors do not encapsulate real time. Instead, we argue that failure detectors encapsulate the fairness in computation and communication. Fairness is a measure of the number of steps executed by one process relative either to the number of steps taken by another process or relative to the duration for which a message is in transit. We argue that partially synchronous systems are perhaps better specified with fairness constraints (rather than real-time constraints) on computation and communication. We demonstrate the utility of this approach by specifying the weakest system models to implement failure detectors in the Chandra-Toueg hierarchy