Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids

Abstract

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.

Evolutionary Engineering of Artificial Proteins with Limited Sets of Primitive Amino Acids

“…It has been suggested that protein solubility is strongly affected by net charge and the fraction of turn-forming residues (Gly, Asp, Pro, Ser and Asn) and is weakly affected by hydrophobicity and protein size (Wilkinson & Harrison, 1991). We found no relation between solubility and the fraction of turn-forming residues, hydrophobicity [calculated based on the index of Kyte and Doolittle (1982)], or protein size for GNN, RNN and NNN proteins (Tanaka et al, 2010). The high solubility of GNN proteins could be attributed to net charge because all GNN proteins lack positively charged amino acids.…”

“…Moreover, targeted proteins with low-copy numbers can be also detected by iterative selection. In the following sections, we describe the application of mRNA display to the construction of random-sequence protein libraries with a limited set of amino acids (Section 4) (Tanaka et al, 2010) and to the selection of functional proteins from partially randomized libraries with a limited set of amino acids (Section 5) (Tanaka et al, 2011). …”

“…Although random-sequence proteins based on three kinds of amino acids (QLR proteins, which consist of Gln, Leu and Arg) tend to strongly aggregate (Davidson & Sauer, 1994;Davidson et al, 1995), random-sequence proteins with the primitive amino acids Ala, Gly, Val, Asp and Glu, which are encoded by codons of the form GNN (N = T, C, A or G), demonstrated extremely high solubility (Doi et al, 2005). Using mRNA display, we constructed three classes of random-sequence libraries consisting of limited sets of amino acids (Tanaka et al, 2010); these libraries were encoded using the codons GNN, RNN (R = A or G, encoding a 12-amino acid alphabet) and NNN (encoding the full set of amino acids). When proteins that were arbitrarily chosen from these libraries were expressed in Escherichia www.intechopen.com coli, all proteins from the GNN library were present in the soluble fraction, all of the proteins from the NNN library were present in the insoluble fraction, and the proteins from the RNN library were intermediate in character, i.e., one out of 14 RNN proteins was expressed only in the soluble fraction, 11 RNN proteins were expressed only in the insoluble fraction, and two were expressed in both fractions (Tanaka et al, 2010).…”

“…Using mRNA display, we constructed three classes of random-sequence libraries consisting of limited sets of amino acids (Tanaka et al, 2010); these libraries were encoded using the codons GNN, RNN (R = A or G, encoding a 12-amino acid alphabet) and NNN (encoding the full set of amino acids). When proteins that were arbitrarily chosen from these libraries were expressed in Escherichia www.intechopen.com coli, all proteins from the GNN library were present in the soluble fraction, all of the proteins from the NNN library were present in the insoluble fraction, and the proteins from the RNN library were intermediate in character, i.e., one out of 14 RNN proteins was expressed only in the soluble fraction, 11 RNN proteins were expressed only in the insoluble fraction, and two were expressed in both fractions (Tanaka et al, 2010). Table 2.…”

“…To investigate this question, we examined the relationship between the solubility of random-sequence proteins and several properties of the amino acid sequences (Tanaka et al, 2010). It has been suggested that protein solubility is strongly affected by net charge and the fraction of turn-forming residues (Gly, Asp, Pro, Ser and Asn) and is weakly affected by hydrophobicity and protein size (Wilkinson & Harrison, 1991).…”