Soluble expression and purification of caspase recruitment domain-containing protein 9

Abstract

Abstract: Objective The caspase recruitment domain-containing protein 9 (CARD9) mediates intracellular signaling by Toll-like and C-type lectin receptors, and plays a critical role in the regulation of fungal infection. Since it is insoluble, the in vitro expression of the complete sequence of CARD9 is currently not feasible; hence, its structure and function remain unclear. In this study, we constructed a prokaryotic systemic expression plasmid using molecular cloning techniques to produce the TrxA-CARD9 protein and its clinical mutant phenotype in vitro. Methods Screening with different isopropyl β-d-1-thiogalactopyranoside concentrations and temperatures led to the successful expression of soluble full-length CARD9. To increase the solubility of the target protein, arginine was used to assist its non-denaturing solubilization. After the removal of nucleic acids and partial heteroprotein using polyethyleneimine and ammonium sulfate precipitation, respectively, the fusion protein was purified using an anion-exchange and heparin affinity chromatography column. Results We successfully achieved highly soluble expression of full-length CARD9 sequence in a prokaryotic system and provided an optimized purification protocol. Conclusion This study provides a basis for future in vitro experiments studying the biological structure and function of CARD9 protein. It proposes a new research idea to find potential targets for the treatment of fungal infections and the treatment of critical fungal infections.

Key words: caspase recruitment domain-containing protein 9, arginine, Trx-A tag, protein expression, insoluble protein

CLC Number:

PENG Min, ZHANG Chen, WANG Jue, CHEN Hao, KONG Qingtao, SANG Hong. Soluble expression and purification of caspase recruitment domain-containing protein 9[J]. Chinese Journal of Mycology, 2023, 18(2): 140-145.

References

[1] HOLLIDAY M J, FERRAO R, de LEON BOENIG G, et al. Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9[J]. J Biol Chem, 2018,293(43):16803-16817.
[2] DRUMMOND R A, FRANCO L M, LIONAKIS M S. Human CARD9:A critical molecule of fungal immune surveillance[J]. Front Immunol, 2018,9:1836. DOI:10.3389/fimmu.2018.01836.
[3] XU Z, QIAO S, QIAN W, et al. CARD9 protects fungal peritonitis through regulating Malt1-mediated activation of autophagy in macrophage[J]. Int Immunopharmacol, 2022,110:108941. DOI:10.1016/j.intimp.2022.108941.
[4] BERTIN J, GUO Y, WANG L, et al. CARD9 is a novel caspase recruitment domain-containing protein that interacts with BCL10/CLAP and activates NF-kappa B[J]. J Biol Chem, 2000,275(52):41082-41086.
[5] QIAO Q, YANG C, ZHENG C, et al. Structural architecture of the CARMA1/Bcl10/MALT1 signalosome:nucleation-induced filamentous assembly[J]. Mol Cell, 2013,51(6):766-779.
[6] GLOCKER E, HENNIGS A, NABAVI M, et al. A homozygous CARD9 mutation in a family with susceptibility to fungal infections[J]. N Engl J Med, 2009,361(18):1727-1735.
[7] ZHONG X, CHEN B, YANG L, et al. Molecular and physiological roles of the adaptor protein CARD9 in immunity[J]. Cell Death Dis, 2018, 9(2):52.
[8] HOLLIDAY M J, WITT A, RODRIGUEZ GAMA A, et al. Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation[J]. Nat Commun, 2019,10(1):3070.
[9] GOEL S, KUEHN H S, CHINEN J, et al. CARD9 expression pattern, gene dosage, and immunodeficiency phenotype revisited[J]. J Clin Immunol, 2022,42(2):336-349.
[10] WOLF E, KIM P S, BERGER B. MultiCoil:a program for predicting two- and three-stranded coiled coils[J]. Protein science, 1997,6(6):1179-1189.
[11] 邓东灵,孔庆涛,胡青原,等. 胱天蛋白酶募集域蛋白9-麦芽糖结合蛋白融合蛋白的原核表达及纯化[J]. 医学研究生学报, 2019,32(3):241-247.
[12] SCHENKEI M, TREFF A, DEBER C M, et al. Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs[J]. Protein Sci, 2021,30(9):1974-1982.
[13] ABE R, KUDOU M, TANAKA Y, et al. Immobilized metal affinity chromatography in the presence of arginine[J]. Biochem Biophys Res Commun, 2009,381(3):306-310.
[14] KIM N A, HADA S, THAPA R, et al. Arginine as a protein stabilizer and destabilizer in liquid formulations[J]. Int J Pharm, 2016,513(1-2):26-37. DOI:10.1016/j.ijpharm.2016.09.003.
[15] HOLLER C, VAUGHAN D, ZHANG C. Polyethyleneimine precipitation versus anion exchange chromatography in fractionating recombinant beta-glucuronidase from transgenic tobacco extract.[J]. J Chromatogr A, 2007,1142(1):98-105.
[16] PRINGELS L, BROECKX V, BOONEN K, et al. Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2018,1089:43-59. DOI:10.1016/j.jchromb.2018.04.045.