Expression of a model peptide of a marine mussel adhesive protein in Escherichia coli and characterization of its structural and functional properties

Kitamura, Masaya; Kawakami, Kiminori; Nakamura, Naotoshi; Tsumoto, Kouhei; Uchiyama, Hidefumi; Ueda, Yoshitaka; Koyanagi, Izumi; Nakaya, Tadao

doi:10.1002/(sici)1099-0518(19990315)37:6<729::aid-pola8>3.0.co;2-3

Cited by 47 publications

(19 citation statements)

References 30 publications

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“…The polarity and adhesive properties increased by hydroxylation process for AdS8. Compared with report, 13 the work of adhesion of hydroxylated AdSP8 were better than those of modified mefp-1 model peptide that are 92.5 mN/m on glass, 67.4 mN/m on PET, and 47.4 mN/m on Teflon.…”

contrasting

confidence: 61%

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

Hatae

Asakura

Fukushima

2007

Polym J

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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...

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contrasting

confidence: 61%

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

Hatae

Asakura

Fukushima

2007

Polym J

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“…Prior to adhesion assays, 1.44 mg of either recombinant Mgfp-5, BSA, or Cell-Tak per ml was modified with 10 Unit of tyrosinase (Sigma) at room temperature for 6 h with shaking. BSA in 5% acetic acid buffer was used as a nonadhesive protein control (2,16). As a positive control, Cell-Tak is a commercially available naturally extracted M. edulis mussel adhesive protein mixture of fp-1 and fp-2 that already contains DOPA residues in 5% acetic acid buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Production of recombinant fp-1 protein has been attempted in several expression systems, including Escherichia coli and yeast (8,22). However, these attempts have failed to express functional and economical mussel adhesive proteins for several reasons, including a highly biased amino acid composition (5 amino acid types comprised ϳ89% of the total amino acids), different codon usage preference between mussel and other expression systems (tRNA utilization problem), and small amount of adhesive produced (16,17,22). Thus, successful mass production technologies have not yet been developed for mussel adhesive proteins.…”

mentioning

confidence: 99%

Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

Hwang¹,

Yoo²,

Jun

et al. 2004

Appl Environ Microbiol

161

125

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Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.

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“…37 Researchers intrigued by this phenomenon have prepared synthetic genes and expressed the corresponding (AKPSYPPTYK) n polypeptides. 38 These model peptides were treated with mushroom tyrosinase to convert the tyrosine units into DOPA, and thereby to induce crosslinking. Because expression yields were low, insufficient material was produced for a thorough evaluation.…”

Section: Mussel Byssus Threadmentioning

confidence: 99%

Protein-based materials, toward a new level of structural control

2001

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Through billions of years of evolution nature has created and refined structural proteins for a wide variety of specific purposes. Amino acid sequences and their associated folding patterns combine to create elastic, rigid or tough materials. In many respects, nature's intricately designed products provide challenging examples for materials scientists, but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes. An increasingly important approach to this problem has been to use biological systems for production of materials. Through protein engineering, artificial genes can be developed that encode protein-based materials with desired features. Structural elements found in nature, such as b-sheets and a-helices, can be combined with great flexibility, and can be outfitted with functional elements such as cell binding sites or enzymatic domains. The possibility of incorporating non-natural amino acids increases the versatility of protein engineering still

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Expression of a model peptide of a marine mussel adhesive protein in Escherichia coli and characterization of its structural and functional properties

Cited by 47 publications

References 30 publications

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

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

Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

Protein-based materials, toward a new level of structural control

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