Litchi thaumatin-like protein (LcTLP) is one of the main causes of the inflammatory response to excessive consumption of litchi in some people.It was proved in the early stage of this study that it had the ability to activate mouse RAW264.7 cells for the activity of inflammatory response to deeply explore the cellular mechanism of inflammation, reveal its crystal structure and biological characteristics such as pro-inflammatory active sites, it was necessary to obtain a large amount of high-purity LcTLP.However, there was no extraction method that could obtain high-purity soluble LcTLP in large quantities in the existing reports.Therefore, the expression vector pCold-NusA-LcTLP was constructed after codon optimization of the LcTLP gene in this study, and the soluble recombinant protein NusA-LcTLP was successfully expressed.Through the two-step purification method of affinity chromatography and gel filtration chromatography, the final yield of recombinant protein could reach 36.26 mg/L, and the purity could reach more than 90%, which showed that this purification scheme could efficiently produce high-purity recombinant LcTLP.After verification of the inflammatory activity of RAW264.7 cells, the recombinant LcTLP stimulated the secretion of nitric oxide to 12.75 μmol/L and 14.68 μmol/L at 1 μg/mL and 2 μg/mL, respectively, which were 2.91 and 3.36 times that of the blank group.It was verified that the recombinant LcTLP had certain pro-inflammatory activity.The expression vector and purification scheme were highly expressed to obtain soluble recombinant LcTLP, which laid the foundation for the subsequent in-depth study of the functional mechanism of LcTLP in cell life activities.
[1] 陈厚彬, 欧良喜, 李建国, 等.新中国果树科学研究70年:荔枝[J].果树学报, 2019, 36(10):1399-1413.
CHEN H B, OU L X, LI J G, et al.Fruit scientific research in New China in the past 70 years:Litchi[J].Journal of Fruit Science, 2019, 36(10):1399-1413.
[2] ZHAO L, WANG K, WANG K, et al.Nutrient components, health benefits, and safety of litchi (Litchi chinensis Sonn.):A review[J].Comprehensive Reviews in Food Science and Food Safety, 2020, 19(4):2139-2163.
[3] PAN M H, ZHU S R, DUAN W J, et al.“Shanghuo” increases disease susceptibility:Modern significance of an old TCM theory[J].Journal of Ethnopharmacology, 2020, 250:112491.
[4] FÄH J, WÜTHRICH B, VIETHS S.Anaphylactic reaction to lychee fruit:Evidence for sensitization to profilin[J].Clinical and Experimental Allergy:Journal of the British Society for Allergy and Clinical Immunology, 1995, 25(10):1018-1023.
[5] HUANG C J, WU M C.Differential effects of foods traditionally regarded as ‘heating’ and ‘cooling’ on prostaglandin E(2) production by a macrophage cell line[J].Journal of Biomedical Science, 2002, 9(6):596-606.
[6] WANG X L, HU X R, YAN H Q, et al.Pro-inflammatory effects of a litchi protein extract in murine RAW264.7 macrophages[J].Horticulture Research, 2016, 3:16017.
[7] CHEN H F, WANG K, XIAO H, et al.Structural characterization and pro-inflammatory activity of a thaumatin-like protein from pulp tissues of Litchi chinensis[J].Journal of Agricultural and Food Chemistry, 2020, 68(23):6439-6447.
[8] MERVAT S M, EL SOUD WALID A, MAGDA F M.Cloning and expression of the recombinant NP24I protein from tomato fruit and study of its antimicrobial activity[J].African Journal of Biotechnology, 2011, 10(65):14276-14285.
[9] 余宇, 王晓杰, 汤春蕾, 等.小麦类甜蛋白TLP基因的克隆与原核表达:中国植物病理学会2007年学术年会[C]. 中国陕西杨凌, 2007.
YU Y,WAMG X J,TANG C, et al.Cloning and prokaryotic expression of a sweet protein like TLP gene from wheat.Chinese Society of Plant Pathology 2007 Annual Conference[C].Yangling, Shaanxi Province, China, 2007.
[10] ZACHARCHENKO T, BARSUKOV I, RIGDEN D J, et al.Biophysical analysis of the N-terminal domain from the human protein phosphatase 1 nuclear targeting subunit PNUTS suggests an extended transcription factor TFIIS-like fold[J].The Protein Journal, 2016, 35(5):340-345.
[11] WANG K, LI W C, WANG K, et al.Litchi thaumatin-like protein induced the liver inflammation and altered the gut microbiota community structure in mice[J].Food Research International, 2022, 161:111868.
[12] DE MATOS L L, STABENOW E, TAVARES M R, et al.Immunohistochemistry quantification by a digital computer-assisted method compared to semiquantitative analysis[J].Clinics, 2006, 61(5):417-424.
[13] 刘素贞, 曹晓敏, 许丽娟, 等.应用CCK8法检测鸡淋巴细胞活性的检测最佳条件研究[J].黑龙江畜牧兽医, 2017(7):212-214.
LIU S Z, CAO X M, XU L J, et al.Application CCK8 chicken method to detect the activity of lymphocyte detection best conditions[J].Heilongjiang Animal Science and Veterinary Medicine, 2017(7):212-214.
[14] MAURO V P.Codon optimization in the production of recombinant biotherapeutics:Potential risks and considerations[J].BioDrugs:Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy, 2018, 32(1):69-81.
[15] 苏惠, 李永光, 谭文雍, 等.大肠杆菌gdhA基因的密码子偏好性分析及优化[J].基因组学与应用生物学, 2015, 34(3):521-529.
SU H, LI Y G, TAN W Y, et al.Analysis of codon usage bias and optimization of codon of gdhA gene in Escherichia coli[J].Genomics and Applied Biology, 2015, 34(3):521-529.
[16] DUMON-SEIGNOVERT L, CARIOT G, VUILLARD L.The toxicity of recombinant proteins in Escherichia coli:A comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3)[J].Protein Expression and Purification, 2004, 37(1):203-206.
[17] LOZANO TEROL G, GALLEGO-JARA J, SOLA MARTÍNEZ R A, et al.Impact of the expression system on recombinant protein production in Escherichia coli BL21[J].Frontiers in Microbiology, 2021, 12:682001.
[18] SUN X M, ZHANG Z X, WANG L R, et al.Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis[J].Biotechnology and Bioengineering, 2021, 118(1):153-163.
[19] UCHINAKA A, HAMADA Y, MORI S, et al.SVVYGLR motif of the thrombin-cleaved N-terminal osteopontin fragment enhances the synthesis of collagen type III in myocardial fibrosis[J].Molecular and Cellular Biochemistry, 2015, 408(1-2):191-203.
[20] 唐威华, 张景六, 王宗阳, 等.SDS-PAGE 法测定His-tag 融合蛋白分子量产生偏差的原因[J].植物生理学报, 2000(1):64-68.
TANG W H, ZHANG J L, WANG Z Y, et al.The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE[J].Acta Phytophysiologica Sinica, 2000(1):64-68.
[21] FUCHS H C, BOHLE B, DALL’ANTONIA Y, et al.Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity[J].Clinical and Experimental Allergy:Journal of the British Society for Allergy and Clinical Immunology, 2006, 36(3):359-368.
[22] WANG L.Characterization of LhSorPR5, a thaumatin-like protein from the oriental hybrid lily sorbonne[J].International Journal of Agriculture and Biology, 2017, 19(4):865-872.
[23] CHA S M, CHA J D, JANG E J, et al.Sophoraflavanone G prevents Streptococcus mutans surface antigen I/II-induced production of NO and PGE2 by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages[J].Archives of Oral Biology, 2016, 68:97-104.
[24] ABARIKWU S O.Kolaviron, a natural flavonoid from the seeds of Garcinia kola, reduces LPS-induced inflammation in macrophages by combined inhibition of IL-6 secretion, and inflammatory transcription factors, ERK1/2, NF-κB, p38, Akt, p-c-JUN and JNK[J].Biochimica et Biophysica Acta (BBA) - General Subjects, 2014, 1840(7):2373-2381.
[25] WANG K, LI W C, WANG K, et al.Structural and inflammatory characteristics of Maillard reaction products from litchi thaumatin-like protein and fructose[J].Food Chemistry, 2022, 374:131821.
