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Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli

  • Applied Genetics and Molecular Biotechnology
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Abstract

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase.

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Acknowledgements

We are grateful to Dr. W. H. Hsu for the critical reading of the manuscript. This work was funded by grants 90AS-2.1.1-FD-Z2 from the Council of Agriculture and NSC89-TSC-7-005-008 from the National Science Council of the Republic of China.

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Authors and Affiliations

  1. Institute of Molecular Biology, National Chung Hsing University, Taichung, 40227, Taiwan, Republic of China

    Yu-Jen Yu, Shih-Cheng Wu, Hung-Hsiang Chan, Yu-Cheng Chen, Zong-Yu Chen & Ming-Te Yang

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  1. Yu-Jen Yu
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  2. Shih-Cheng Wu
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  3. Hung-Hsiang Chan
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  4. Yu-Cheng Chen
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Correspondence to Ming-Te Yang.

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Fig. S1

Nucleotide sequence and deduced amino acid sequences of the 1,535-bp DNA fragment containing the S. netropsis TGase gene. Numbers on the right side denote nucleotides and the deduced amino acid sequences, respectively. The putative ribosome-binding site (RBS) is shown on a gray background and underlined. The putative signal peptide and proregion are indicated by thin and thick underlining, respectively. The N-terminal amino acid sequence obtained from mature Sn TGase is shown in boldface. The asterisk represents the stop codon (DOC 33 KB)

Fig. S2

Schematic representation of the Sn TGase (prepro, pro, and mature), recombinant Sn proTGase, and processed mature TGase constructs. The site of cleavage of the recombinant proTGase by bovine trypsin is indicated by an arrow (DOC 121 KB)

Fig. S3

Processing of the recombinant Sn proTGase by proteases in the culture broth of S. netropsis. Cell-free culture broth of S. netropsis was collected after 20 h of cultivation. Ten micrograms of the purified recombinant Sn proTGase was added to 400 μL of the culture broth and the reaction was carried out at 37°C for 3 h. In vitro processing of the recombinant Sn proTGase was monitored by SDS-PAGE analysis. Lane 1 purified recombinant Sn Trx–proTGase, lane 2 culture supernatant of S. netropsis, lane 3 purified recombinant Sn Trx–proTGase was added to the culture supernatant of S. netropsis, lane 4 purified processed mature TGase (DOC 82.5 KB)

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Yu, YJ., Wu, SC., Chan, HH. et al. Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli . Appl Microbiol Biotechnol 81, 523–532 (2008). https://doi.org/10.1007/s00253-008-1688-7

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  • DOI: https://doi.org/10.1007/s00253-008-1688-7

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