Enhanced Production of a Thermostable Carbonic Anhydrase in by Using a Modified NEXT Tag.

Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO capture and utilization. In particular, the CA of (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins.