Improving production yield of 3,4-Dihydroxyphenylalanine-incorporated recombinant mussel adhesive protein from E. coli

Abstract

Mussels secrete a variety of adhesive proteins, which are considered as potential water-resistant bioglue. The key for strong adhesion of mussel adhesive protein (MAP) is the amino acid analogue with a reactive catechol group called 3,4 –Dihydroxyphenylalanine (DOPA). The catechol group not only forms hydrogen bond to silicon or metal ions, also oxidized to DOPA-Quinone so the proteins cross-link each other. As MAPs are harmless and do not cause immunological responses to human, they could be applied for medical glue. Although the tyrosinase in mussels transforms tyrosine to DOPA under natural condition, extracting the proteins from mussels is labor-intensive and needs high production cost. Therefore, many research groups have been studied the recombinant MAP (rMAP) in bacteria or yeast. Yang and their colleagues showed that rMAP production in auxotrophic Escherichia coli (E. coli) under minimal condition had high incorporation yield of DOPA over 90% by endogenous tyrosyl-tRNA synthetase. However, the yield of purified rMAP was low, around 3-5mg/L. In order to commercialize the rMAP as a bioglue, it is necessary to improve the yield of purified rMAP in E. coli. In this study, we developed the translational machinery in E.coli to improve production yield of rMAP with a high DOPA modification yield. We designed the dual vector system using one vector for DOPA specific aminoacyl tRNA synthetase (mut MjTyrRS) with Mj tRNA_(AUA ) and the other for rMAP. Also, for overexpression of E.coli tyrosyl tRNA synthetase (TyrRS), we designed E.coli TyrRS and rMAP in each plasmid. We examined improvement of production yield and high DOPA content in rMAP by the development of translational machinery. This study supports that rMAP has high DOPA content and high production rates at the same time so that it can be commercialized as a bioglue.