High-level Recombinant Expression and Refolding of Lysozyme from the Marine Shrimp Marsupenaeus japonicus
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Shrimp lysozyme is as an antibacterial enzyme that participates in the innate defense against the invasion of bacterial pathogens. In this study, the lysozyme gene from hemocytes of the shrimp Marsupenaeus japonicus was isolated and characterized. The M. japonicus lysozyme (MjLys) encodes a polypeptide of 158 amino acids (aa) that includes an 18 aa signal peptide. The gene fragment encoding the mature MjLys protein was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS, and the protein was strongly expressed in insoluble inclusion bodies. Following extraction using urea, the denatured recombinant protein was refolded by on-column Ni2+ affinity chromatography or dialysis with a gradient of decreasing urea concentration. Approximately 50% of the recombinant MjLys was successfully refolded into monomeric protein using urea gradient dialysis, while 30% was salvaged using on-column refolding. Purified MjLys exhibited significant antibacterial activity against Gram-positive bacteria Micrococcus lysodeikticus and Staphylococcus aureus. This efficient over-expression and refolding method can provide the large quantities of biologically active protein required for further biochemical and structural studies and potential biotechnological applications.
KeywordsCrustaceans Marsupenaeus japonicus c-type lysozyme recombinant expression
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- 10.Gross, P.S., Bartlett, T.C., Browdy, C.L., et al., Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the pacific white shrimp, Litopennaeus vannamei, and the atlantic white shrimp, L. setiferus, Dev. Comp. Immunol., 2001, vol. 25, no. 7, pp. 565–577.Google Scholar
- 15.Liu, M., Zhu, K.L., Li, H.B., et al., Bacterial community composition of the Yellow Sea Cold Water Mass studied by PCR-denaturing gradient gel electrophoresis, J. Environ. Sci. (Beijing, China), 2008, vol. 29, no. 4, pp. 1082–1091.Google Scholar
- 20.Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory, 2001.Google Scholar