(78h) Evolution of Inteins for Mammalian Cell Expression

The lack of a platform technology for the purification of non-mAb proteins remains a major concern in the biopharmaceutical industry.  The implementation of the Protein A platform for the purification of monoclonal antibodies has revolutionized the industry.  However, no analogous purification platform exists for non-antibody products.  Purification tags present one intriguing possibility for the development of such a platform.  The use of purification tags is ubiquitous at small scale; however, the cost of tag removal becomes prohibitive upon scale-up.  To address this, we have developed self-cleaving purification tags based on intein technology.  This intein based system has been well established for expression and purification of proteins in E. coli.  The major limitation of the technology is due to premature cleavage; intein cleavage during expression leads directly to product losses.  Due to premature cleavage, the technology is currently limited to bacterial expression systems.  The goal of the current work is to develop inteins that are suitable for use in mammalian cell expression systems.

The current work describes the development of inteins suitable for mammalian cell expression via directed evolution.  We have developed a high throughput screen for intein evolution based on yeast surface display.  We have combined this screen with a targeted library of intein mutants to select for improved variants.  We report progress toward the development of inteins with a shifted pH optimum for cleaving and increased sensitivity to small molecule inhibition that can be used in mammalian cell expression systems.  This would represent a significant advance in the purification of non-mAb proteins for the biopharmaceutical industry.