<i>In vivo</i> post‐translational modifications of recombinant mussel adhesive protein in insect cells

Lim, Seonghye; Kim, Kyoung Ro; Choi, Yanghee; Kim, Dae-Kyum; Hwang, Daehee; Joon, Hyung

doi:10.1002/btpr.662

Cited by 28 publications

(20 citation statements)

References 40 publications

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“…These results demonstrated that tyrosines at position 5 in the decapeptide unit of the fp-1 region in the co fp-151 were in vivo modified into Dopaquinone through co-expression of tyrosinase, although the modification of tyrosine residues was more frequently detected at position 9 than position 5 in the natural fp-1 [25]. Previously, the tyrosine modification at position 5 was observed in synthetic fp-1 decapeptide conversion using mushroom tyrosinase [9] and was also observed with in vivo modification of fp-151 in insect Sf9 cells [26]. Thus, the altered preference of hydroxylation position might be due to the different modification enzymes and/or the different structural features of fp-151.…”

Section: Resultsmentioning

confidence: 99%

In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

Choi

Yang

et al. 2012

Microb Cell Fact

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BackgroundIn nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo.ResultsA recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151.ConclusionHere, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in practical applications and can be successfully applied to prepare other Dopa/Dopaquinone-based biomaterials.

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Section: Resultsmentioning

confidence: 99%

In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

Choi

Yang

et al. 2012

Microb Cell Fact

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“…In addition, many native protein-based materials incorporate repetitive sequences that include multiple sites of post-translational modification that are critical for appropriate biological function, e.g., trans -hydroxyproline in collagens, [52–53] or DOPA residues in mussel adhesive proteins. [54] The multi-site suppression techniques represents an attractive approach to the synthesis of these functional materials, which are challenging to obtain from in vitro chemical or in vivo enzymatic modification of the native protein sequences.…”

Section: Resultsmentioning

confidence: 99%

Multiple Site‐Selective Insertions of Noncanonical Amino Acids into Sequence‐Repetitive Polypeptides

Wu¹,

Patterson²,

Desai³

et al. 2013

ChemBioChem

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A simple and efficient method is described for introduction of non-canonical amino acids at multiple, structurally defined sites within recombinant polypeptide sequences. E. coli MRA30, a bacterial host strain with attenuated activity for release factor 1 (RF1), is assessed for its ability to support the incorporation of a diverse range of non-canonical amino acids in response to multiple encoded amber (TAG) codons within genetic templates derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and isolated protein yield were observed to depend on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNACUA pair and the non-canonical amino acid substrate. This approach afforded elastin-mimetic protein polymers containing non-canonical amino acid derivatives at up to twenty-two positions within the repeat sequence with high levels of substitution. The identity and position of the variant residues was confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments resulting from thermolysin digestion. The accumulated data suggest that this multi-site suppression approach permits the preparation of protein-based materials in which novel chemical functionality can be introduced at precisely defined positions within the polypeptide sequence.

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“…Compared to other mussels species, A. ater contains 15-20 times more adhesive polyphenolic proteins than any other mussels [72]. Protocols aimed at optimizing large scale production of these biological adhesive for their industrial usage have recently been developed and may accelerate the use of such bioinspired proteins in various biomedical and tissue engineering fields [73,74].…”

Section: Economical Perspectivementioning

confidence: 99%

Biomonitoring Climate Change and Pollution in Marine Ecosystems: A Review onAulacomya ater

Caza

Cledón

St‐Pierre

2016

Journal of Marine Biology

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The sedentarism and wide global distribution of the blue musselMytilus edulishave made it a useful bioindicator to assess changes in the health status of the marine ecosystem in response to pollution and other environmental stresses. Effective biomonitoring of an ecosystem requires, however, that multiple biomarkers be used to obtain an accurate measure of the cumulative effects of different sources of environmental stress. Here, we provide a first integrated review of the biological, economical, and geographical characteristics of another species of mussels, the ribbed musselAulacomya ater. We discuss the use ofAulacomya ateras a complementary biomonitor to the blue mussel to assess the impact of pollutants and climate change. Recent findings have indeed shown thatMytilus edulisandAulacomya aterhave distinctive anatomy and physiology and respond differently to environmental stress. Monitoring of mixed beds containing blue and ribbed mussels may thus represent a unique opportunity to study the effect of environmental stress on the biodiversity of marine ecosystems, most notably in the Southern hemisphere, which is particularly sensitive to climate change and where both species often cohabitate in the same intertidal zones.

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In vivo post‐translational modifications of recombinant mussel adhesive protein in insect cells

Cited by 28 publications

References 40 publications

In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

Multiple Site‐Selective Insertions of Noncanonical Amino Acids into Sequence‐Repetitive Polypeptides

Biomonitoring Climate Change and Pollution in Marine Ecosystems: A Review onAulacomya ater

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