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Recombinant Production of Crab Antimicrobial Protein Scygonadin Expressed as Thioredoxin and SUMO Fusions in Escherichia coli

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Abstract

Scygonadin is an antimicrobial protein isolated from the mud crab, Scylla serrate. The mature protein comprises 102 amino acids and has a theoretical molecular weight of 11,272 Da. The protein’s specific expression pattern strongly suggests that it plays a role in reproductive immunity. In this study, I developed a protocol for producing recombinant scygonadin in Escherichia coli. The target protein was expressed as both thioredoxin and SUMO fusions, and released by TEV and SUMO protease-mediated cleavages, respectively. In either case, the liberated scygonadin was separated from its carrier using a HisTrap HP column. From thioredoxin and SUMO fusion constructs, 32.7 and 29.2 mg target protein per liter of culture was obtained, respectively. The described protocol provides an effective means for producing scygonadin in relatively large quantities, which facilities its further characterization.

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Acknowledgments

This work was supported by departmental funding dedicated to the protein production core facility. The author is grateful to Dr. Altenbuchner (Universität Stuttgart, Germany) for plasmids pJOE4905.1 (which encodes MBP and SUMO dual-tagged GFP) and pJOE4847.2 (which encodes MBP tagged SUMO protease Ulp1). The author would like to thank Sammy Pardo for mass spectrometry analysis.

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  1. Protein Production Core Facility, Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA

    Yifeng Li

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  1. Yifeng Li
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Correspondence to Yifeng Li.

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Li, Y. Recombinant Production of Crab Antimicrobial Protein Scygonadin Expressed as Thioredoxin and SUMO Fusions in Escherichia coli . Appl Biochem Biotechnol 169, 1847–1857 (2013). https://doi.org/10.1007/s12010-013-0102-9

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  • DOI: https://doi.org/10.1007/s12010-013-0102-9

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