In vivo and in vitro characterization of hydrophilic protein tag-fused Ralstonia eutropha polyhydroxyalkanoate synthase.

This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaCRe) by fusing a hydrophilic tag, glutathione S-transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaCRe. However, an in vitro assay revealed that the surface of GST-fused PhaCRe (GST-PhaCRe) had increased hydrophilicity, and tended to form correct PhaCRe dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaCRe displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaCRe showed higher catalytic turnover than PhaCRe in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaCRe. PMID: 31315020 [PubMed - as supplied by publisher]
Source: International Journal of Biological Macromolecules - Category: Biochemistry Authors: Tags: Int J Biol Macromol Source Type: research