Prokaryotic expression of Cryptococcus neoformans laccase

Abstract

Abstract:

Objective To construct a prokaryotic expression vector of Cryptococcus neoformans laccase containing His-tag and to express and identify it.Methods The coding sequence of LAC1 gene was amplified by PCR and correctly inserted into pET-28a (+) vector to obtain the recombinant plasmid. The recombinant plasmid was transformed into E.coli DH5α competent cells for PCR identification and gene sequencing. The correct recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. Induction expression was carried out under different conditions, and the target protein was detected and identified by SDS-PAGE electrophoresis and MALDI-TOF mass spectrometry. Inclusion body protein was denatured by urea and identified by SDS-PAGE electrophoresis and MALDI-TOF mass spectrometry.Results The prokaryotic expression vector of Cryptococcus neoformans laccase containing His-tag was constructed and confirmed by PCR, and the result of sequencing was consistent with the target sequence. The SDS-PAGE showed that the target protein with molecular mass(MR) about 68 000 was successfully induced to express in E. coli BL21(DE3). And identification by MALDI-TOF mass spectrometry demonstrated that the target protein is insoluble in this expression system and may exist in the form of inclusion body. Conclusion The prokaryotic expression vector of Cryptococcus neoformans laccase containing His-tag was successfully constructed. This laid the foundation for further purification and analysis of the crystal structure of Cryptococcus neoformans laccase.

Key words: Cryptococcus neoformans, laccase, prokaryotic expression, inclusion protein

CLC Number:

R379.5

DENG Dong-ling, KONG Qing-tao, ZHANG Yuan-yuan, YUAN Hong-shan, LIU Hui, SANG Hong. Prokaryotic expression of Cryptococcus neoformans laccase[J]. Chinese Journal of Mycology, 2018, 13(4): 208-212.

References

[1] Heitman J, Kozel TR, Kwonchung KJ, et al. Cryptococcus:from human pathogen to model yeast[J]. Lancet Infect Dis, 2011, 11(6):434-434.
[2] Kelliher CM, Haase SB. Connecting virulence pathways to cell-cycle progression in the fungal pathogen Cryptococcus neoformans[J]. Curr Genet, 2017, 63(5):803-811.
[3] Kwonchung KJ, Polacheck I. Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice[J]. J Bacteriol, 1982,150(3):1414-1421.
[4] Hamilton AJ, Gomez BL. Melanins in fungal pathogens[J]. J Med Microbiol, 2002, 51(3):189-191.
[5] Almeida F, Wolf JM. Virulence-associated enzymes of Cryptococcus neoformans[J].Eukaryotic cell,2015,14(12):1173-1185.
[6] Mohagheghpour N, Waleh N, Garger SJ, et al. Synthetic melanin suppresses production of proinflammatory cytokines[J]. Cell Immunol, 2000, 199(1):25-36.
[7] Nosanchuk JD, Stark RE. Fungal melanin:What do we know about structure[J]? Front Microbiol, 2015, 6:1463.
[8] Williamson PR. Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans:identification as a laccase[J]. J Bacteriol, 1994, 176(3):656.
[9] Rodgers CJ, Blanford CF, Giddens SR, et al. Designer laccases:a vogue for high-potential fungal enzymes[J]?Trends Biotechnol. 2010, 28(2):63-72.
[10] 陈映丹, 陆春, 冯佩英. 黑素与真菌抗药性的研究进展[J]. 中国真菌学杂志, 2016, 11(4):248-251.
[11] Cannatelli MD, Ragauskas AJ. Two decades of laccases:advancing sustainability in the chemical industry[J]. Chemical Record, 2017, 17(1):122-140.
[12] Missall TA, Moran JM, Corbett JA, et al. Distinct stress responses of two functional laccases in Cryptococcus neoformans are revealed in the absence of the thiol-specific antioxidant Tsa1[J]. Eukaryotic Cell, 2005, 4(1):202-208.
[13] Ducros V, Brzozowski AM, Wilson KS, et al. Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 A resolution[J]. Nat Struct Biol, 1998, 5(4):310-316.
[14] Benjamin S, Anna S, Ernst H. Use of anionic denaturing detergents to purify insoluble proteins after overexpression[J]. BMC Biotechnol, 2012, 12(1):95.
[15] Zhao Q, Wei D, Li Z, et al. GWT1 encoding an inositol acyltransferase homolog is required for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans[J]. FEMS Yeast Res, 2015, 15(8). pii:fov086.
[16] Garcíarodas R, Trevijanocontador N, Román E, et al. Role of Cln1 during melanization of Cryptococcus neoformans[J]. Front Microbiol, 2015, 6:798.