Optimization of a recombinant BlaR-CTD protein formulation using the Response Surface Methodology

Abstract

A penicillin-binding protein (PBP) is a group of proteins that have an affinity for and bind to penicillin. They are a normal component of many bacteria and play a crucial role in bacterial cell wall synthesis. PBPs are the targets of β-lactam antibiotics, such as penicillin. The sequence of a carboxy-terminal of the β-lactam sensor-transducer protein (BlaR-CTD) from Bacillus licheniformis ATCC14580 was extracted from US7745193B2 patent and expressed in E. coli using pColdI vector as a soluble His-tag recombinant protein. In this study, several excipients were used to improve the stability of recombinant BlaR-CTD and obtain the optimal formulation for this protein using response surface methodology (RSM)/ Central Composite Design (CCD). Total protein concentration was measured by UV spectroscopy and Bradford test. A total of 7 various formulations were designed using four different excipients including Glycerol, Sucrose, Triton x-100 and Tween-20, and three different buffers like Tris, Borate and PBS. By obtaining suitable excipients and buffer i.e. glycerol and sucrose, several concentrations of pH 7 to 9 were evaluated. The pH 7.62, glycerol 15.35% and sucrose 152.52 mM were determined as the most suitable for improve thermal stability of recombinant BlaR-CTD.