Biosynthesis and Characterization of the Artificial Protein Consisting of Marine Mussel Adhesive Protein and Silk-Like Protein Sequences

Abstract

Mussel Mytilus edulis attach to solid surfaces in the turbulent environment by making a byssus, which consists of several redox-active proteins. 1 The first of these proteins to be characterized from the mussel M. edulis foot protein 1 (mefp-1) was found to consist of predominantly repeated decapeptide sequences. 2,3 The most frequently repeated sequence is AKPSYP Ã P ÃÃ TY Ã K, where P Ã , P ÃÃ and Y Ã denote trans-2,3-cis-3,4-dihydroxyproline (diHyp), 4-hydroxyproline (Hyp), and 3,4-dihydroxyphenylalanine (Dopa), respectively.4,5 The Dopa residues are believed to result from the enzyme reaction of a catechol oxidase, which converts tyrosine residues to Dopa and subsequently to ortho-quinones. 6 The Dopa residues are key components that are believed to be primarily responsible for chemisorption of the adhesion proteins to substrates underwater. 7 The ortho-quinone residues can react to lysine residues, which are active component of covalent cross-linking of the adhesive. The adhesive ability is influenced by secondary structure and increased with higher -structure content. 8 We attempted that the Bombyx mori silk fibroin GAGAGS repetitive sequence 9 was conjugated to the mefp-1 sequence in order to construct a novel adhesive protein with high -structure content. Conjugated sequence GAGAGS is frequently found in the -structure domain of silk fibroin. It was reported that a byssus contained fiber protein consisting of predominantly repeated sequence that combine collagen with flanking domains that resemble silk-fibroin or elastin.10 But the fiber protein is one-component protein of byssus and is not conjugated to mefp-1 sequence. In this study, we attempted to construct the protein that is more adhesive materials than mefp-1 by introduction of higher -structure, silk-sequence.The novel adhesive protein was designed repetitive motif sequence, which is TS(AKPSYPPTYK) 2 (GAGAGS) 2 AS (AdSP1). The amino acid Pro and Tyr are the original amino acids of diHyp, Hyp and Dopa before post-translational modification, which are used instead of diHyp, Hyp and Dopa for AdSP.Herein we describe the biosynthesis of 8 repeats AdSP polymer (AdSP8) by genetically engineered technique, and characterization of AdSP8.AdSP8 was expressed as the fusion protein with terminal regions of histidine tagged sequence. The terminal regions can be removed by cyanogen bromide (CNBr) cleavage at flanking methionine residues. The target protein and the expressed proteins can be represented in Scheme 1. All recombinant techniques were used in general method and the polymerization of the DNA was accomplished by head-to-tail construction strategy method.11 Protein expression was performed under control of T7 promoter. pET-30a and BL21(DE3)pLysS from Novagen were used as expression vector and host, respectively.The expressed protein was purified by nickel chelate affinity-chromatography by stepwise concentration of imidazole with 3 M Urea buffer.Cleavage of the expressed protein was accomplished in peculiar condition of CNBr cleavage. Forty-five mg of e...