Small design from big alignment: engineering proteins with multiple sequence alignment as the starting point.

Multiple sequence alignment (MSA) is a fundamental way to gain information that cannot be obtained from the analysis of any individual sequence included in the alignment. It provides ways to investigate the relationship between sequence and function from a perspective of evolution. Thus, the MSA of proteins can be employed as a reference for protein engineering. In this paper, we reviewed the recent advances to highlight how protein engineering was benefited from the MSA of proteins. These methods include (1) engineering the thermostability or solubility of proteins by making it closer to the consensus sequence of the alignment through introducing site mutations; (2) structure-based engineering proteins with comparative modeling; (3) creating paleoenzymes featured with high thermostability and promiscuity by constructing the ancestral sequences derived from multiple sequence alignment; and (4) incorporating site-mutations targeting the evolutionarily coupled sites identified from multiple sequence alignment.