High solubility of random-sequence proteins consisting of five kinds of primitive amino acids

Searching for functional proteins among random-sequence librariesis a major challenge of protein engineering; the difficultiesinclude the poor solubility of many random-sequence proteins.A library in which most of the polypeptides are soluble andstable would therefore be of great benefit. Although modernproteins consist of 20 amino acids, it has been suggested thatearly proteins evolved from a reduced alphabet. Here, we haveconstructed a library of random-sequence proteins consistingof only five amino acids, Ala, Gly, Val, Asp and Glu, whichare believed to have been the most abundant in the prebioticenvironment. Expression and characterization of arbitrarilychosen proteins in the library indicated that five-alphabetrandom-sequence proteins have higher solubility than do 20-alphabetrandom-sequence proteins with a similar level of hydrophobicity.The results support the reduced-alphabet hypothesis of the primordialgenetic code and should also be helpful in constructing optimizedprotein libraries for evolutionary protein engineering.