Department of Biosciences and Informatics, Keio University, Yokohama 223-8522, Japan.
Protein Sci. 2010 Apr;19(4):786-95. doi: 10.1002/pro.358.
Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279-284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids.
从随机序列文库中筛选功能蛋白已被用于进化蛋白质工程领域的新型蛋白质的进化。然而，由 20 种天然氨基酸组成的随机序列蛋白往往会聚集，并且随机序列文库中功能蛋白的出现率很低。从生命起源的角度来看，有人提出原始蛋白由一组有限的氨基酸组成，这些氨基酸在化学进化的早期可能大量形成。我们之前发现，由 5 种原始氨基酸构建的随机序列蛋白文库中的成员表现出很高的溶解性（Doi 等人，Protein Eng Des Sel 2005；18:279-284）。尽管这样的文库有望找到功能蛋白，但功能可能有限，因为它们没有带正电荷的氨基酸。在这里，我们使用 mRNA 显示（消除包含终止密码子和移码的序列）进行预选，构建了由 5、12 和 20 种氨基酸组成的三个 120 个氨基酸随机序列蛋白文库，并对这些文库中任意选择的随机序列蛋白的结构特性进行了表征和比较。我们发现，用 12 个字母（包括 5 个原始氨基酸和带正电荷的氨基酸）构建的随机序列蛋白的溶解性高于用 20 个字母构建的随机序列蛋白，尽管这两个文库中的其他生物物理特性非常相似。因此，与用 20 种氨基酸构建的文库相比，由 12 种氨基酸构建的中等复杂度文库可能更适合功能筛选。
