1. Cloning and high-level SUMO-mediated fusion expression of a serine protease inhibitor from Hyphantria cunea Drury that exhibits activity against papain.
- Author
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Sang, Ming, Xu, Chen, Wei, Zhiheng, Wu, Xiaolong, Guo, Yuxing, Li, Jianfeng, Wang, Zhiguo, and Zhang, Jiaxin
- Subjects
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ELASTASES , *PROTEASE inhibitors , *CHIMERIC proteins , *CATHEPSIN B - Abstract
Abstract Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli , resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2–12), temperature (20–50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure. Highlights • The full length dHC-serpin gene cloned from Hyphantria cunea Drury. • The SUMO-dHC-serpin fusion protein is highly soluble in Escherichia coli. • dHC-serpin exhibits activity against papain. • dHC-serpin remained active and stable between 20 °C and 50 °C and pH 2 and 12. • The reactive center loop region is important for dHC-serpin binding with papain. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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