One of the primary goals of protein design is to engineer proteins with improved stability. Protein stability is a key issue for chemical, biotechnology and pharmaceutical industries. The development of robust proteins/enzymes with the ability to withstand the potentially harsh con- ditions of industrial operations is of high importance. A number of strategies are currently being employed to achieve this goal. Two particular approaches, (i) directed evolution and (ii) computational protein design, are quite powerful yet have only recently been combined or applied and analyzed in parallel. In directed evolution, libraries of variants are searched experimentally for clones possessing the desired properties. With computational methods, protein design algorithms are utilized to perform in silico screening for stable protein sequences. Here, we used gene libraries of an unstable variant of streptococcal protein G (Gβ1) and an in vivo screening method to identify stabilized variants. Many variants with notably increased thermal stabilities were isolated and characterized. Concomitantly, computational techniques and protein design algorithms were used to perform in silico screening of the same destabilized variant of Gβ1. The combined use, and critical analysis, of these methods promises to advance the field of protein design.
Submitter: Marie Ary
Submission Date: July 31, 2017, 11:46 a.m.
|Number of data points
|Experimental Assay: Bacterial growth in 3000 ug/ml carbenicillin (antibiotic) ; Experimental Assay: Tm ; Computational Protocol: ORBIT total energy
|in vivo chimeric screen of saturation mutagenesis lib compared to GMEC from CPD at positions 23, 27, 45