Combined use of experimental and computational screens to characterize protein stability.
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 conditions 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 (Gbeta1) 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 Gbeta1. The combined use, and critical analysis, of these methods promises to advance the field of protein design.
Barakat
Nora H
NH
Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Dr, San Diego, CA 92182-1030, USA.
Barakat
Nesreen H
NH
Love
John J
JJ
D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D019295:Computational Biology / Q000379:methods*; D019143:Evolution, Molecular; D015723:Gene Library; D009154:Mutation; D011489:Protein Denaturation; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis; D013696:Temperature
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011506:Proteins