LwaCas13a蛋白的表达纯化与活性分析

doi:10.16431/j.cnki.1671-7236.2023.07.034

Abstract

Abstract: 【Objective】 The production collection of high-purity, high-concentration LwaCas13a protein capable of excellent trans-cleavage activity from Leptotrichia wadei was undertaken in vitro, which could provide an important biological tool for the development of new methods in animal virus RNA detection based on CRISPR-LwaCas13a system.【Method】 The recombinant plasmid pET15b-SUMO-LwaCas13a was constructed by PCR, double digestion as well as ligand reaction, and prokaryotic expression and condition optimization were performed.Ferment 100 mL of Escherichia coli BL21(DE3) was used to obtain the expression product and assess the purification procedure, the induced expression and purified products were concentrated by ultrafiltration, and the protein concentration was detected by BCA.The purified LwaCas13a protein was co-incubated with CRISPR RNA (CrRNA), target RNA from Porcine epidemic diarrhea virus (PEDV) and fluorescent-labeled single stranded RNA (ssRNA).A full-wavelength fluorescence analyzer was used to verify the trans-cleavage activity of CRISPR-LwaCas13a, which was also compared with commercial Cas13a protein.The optimal binding ratio between LwaCas13a and CrRNA was optimized for further research.【Result】 The recombinant plasmid pET15b-SUMO-LwaCas13a was successfully constructed.The optimal IPTG concentration of LwaCas13a recombinant protein was 0.4 mmol/L, and the optimum induction temperature and time was 18 ℃ for 16 h.After a series of purifications, LwaCas13a protein was produced at a purity of 95% and concentration of 7.5 mg/mL.The purified LwaCas13a protein was verified to have readily detectable trans-cleavage activity of ssRNA probes, and found its trans-cleavage activity was better than that of commercial Cas13a protein by about 1.25 times at the same concentration.The optimal binding ratio between LwaCas13a protein and CrRNA was 1:2.【Conclusion】 In this study, LwaCas13a protein at high purity and concentration was successfully obtained, and the purified LwaCas13a had excellent trans-cleavage activity than commercial Cas13a in vitro with the optimal binding ratio between LwaCas13a and CrRNA, which provided a basis for the development and use of CRISPR-LwaCas13a for animal virus RNA detection.

Key words: LwaCas13a protein; prokaryotic expression; SUMO enzyme digestion; trans-cleavage activity

CLC Number:

S852.65

DOU Ling, LI Zexuan, JIA Hexiao, ZHANG Caiyun, ZUO Fujiang, LI Ting, XIE Yuanjiang, DUAN Xiaolei. Expression, Purification and Activity Analysis of LwaCas13a Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2941-2950.

References

[1] LIU G, LIN Q, JIN S, et al.The CRISPR-Cas toolbox and gene editing technologies[J].Molecular Cell, 2022, 82(2):333-347.
[2] RODOLPHE BARRANGOU.The roles of CRISPR-Cas systems in adaptive immunity and beyond[J].Current Opinion in Immunology, 2015, 32:36-41.
[3] PICKAR-OLIVER A, GERSBACH C A.The next generation of CRISPR-Cas technologies and applications[J].Nature Reviews Molecular Cell Biology, 2019, 20(8):490-507.
[4] COX D B T, GOOTENBERG J S, ABUDAYYEH O O, et al.RNA editing with CRISPR-Cas13[J].Science, 2017, 358(6366):1019-1027.
[5] EASAT-SELETSKY A, O'CONNELL M R, KNINGHT S C, et al.Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection[J].Nature, 2016, 538(7624):270-273.
[6] SHIHONG GAO D, ZHU X, LU B.Development and application of sensitive, specific, and rapid CRISPR-Cas13-based diagnosis[J].Journal of Medical Virology, 2021, 93(7):4198-4204.
[7] 辛忠昊, 郭效珍, 逯晓寒, 等.猪流行性腹泻病毒变异毒株的分离鉴定及遗传演化分析[J].生物学杂志, 2022, 39(3):12-17. XIN Z H, GUO X Z, LU X H, et al.Isolation and identification of variant strains of Porcine epidemic diarrhea virus and analysis of genetic evolution[J].Journal of Biology, 2022, 39(3):12-17.(in Chinese)
[8] 左媛媛, 徐天刚, 戈胜强, 等.我国猪流行性腹泻病毒流行现状及分子遗传演化特征[J].中国动物检疫, 2022, 39(9):9-17. ZUO Y Y, XU T G, GE S Q, et al.Prevalence and molecular genetic evolution of Porcine epidemic diarrhea virus in China[J].China Animal Health Inspection, 2022, 39(9):9-17.(in Chinese)
[9] JUONG J, LADHA A, SAITO M, et al.Detection of SARS-CoV-2 with SHERLOCK one-pot testing[J]. New England Journal of Medicine, 2020, 383(15):1492-1494.
[10] YA F C, YUE D, TIAN Y L, et al.Visual detection of Porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a[J].Transboundary and Emerging Diseases, 2020, 67(2):564-571.
[11] 刘茹, 李小龙, 张晓倩, 等.LbCas12a蛋白的原核表达及活性检测[J].中国畜牧兽医, 2022, 49(5):1621-1629. LIU R, LI X L, ZHANG X Q, et al.Prokaryotic expression and activity identification of LbCas12a protein[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(5):1621-1629.(in Chinese)
[12] LIU L, LI X, MA J, et al.The molecular architecture for RNA-guided RNA cleavage by Cas13a[J].Cell, 2017, 170(4):714-726.e10.
[13] 曹汝菲, 李泽轩, 许欢, 等.脆弱拟杆菌Pif1解旋酶的表达纯化与晶体生长[J].生物技术通报, 2021, 37(9):180-190. CAO R F, LI Z X, XU H, et al.Expression, purification, and pcrystallization of Pif1 helicase from Bacteroides fragilis[J]. Biotechnology Bulletin, 2021, 37(9):180-190.(in Chinese)
[14] IELKOPF C L, BAUER W, URBATSCH I L.Methods for measuring the concentrations of proteins[J].Cold Spring Harbor Protocols, 2020, 2020(4):102277.
[15] 王勋, 张雨杭, 孙亚宁, 等.CRISPR/Cas13a重组蛋白表达纯化及其连带剪切酶活性的鉴定[J].河南农业科学, 2021, 50(4):147-153. WANG X, ZHANG Y H, SUN Y N, et al.Expression and purification of CRISPR/Cas13a and establishment of its collateral RNase activity assay[J].Journal of Henan Agricultural, 2021, 50(4):147-153.(in Chinese)
[16] LOTFI M, REZAEI N.CRISPR/Cas13:A potential therapeutic option of COVID-19[J].Biomedicine & Pharmacotherapy, 2020, 131:110738.
[17] WANG Y, ZHANG Y, CHEN J, et al.Detection of SARS-CoV-2 and its mmutated variants via CRISPR-Cas13-based transcription amplification[J].Analytical Chemistry, 2021, 93(7):3393-3402.
[18] ZHANG K, ZHANG Z, KANG J, et al.CRISPR/Cas13 d-mediated microbial RNA knockdown[J].Frontiers in Bioengineering and Biotechnology, 2020, 8:856.
[19] ABUDAYYEH O O, GOOTENBERG J S, ESSLETZBICHLER P, et al.RNA targeting with CRISPR-Cas13[J].Nature, 2017, 550(7675):280-284.
[20] KNOTT G J, EAST-SELETSKY A, COFSKY J C, et al.Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme[J].Nature Structural & Molecular Biology, 2017, 24(10):825-833.
[21] MALAKAR P, VENKATESH K V.Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins[J].Applied Microbial and Cell Physiology, 2012, 93(6):2543-2549.
[22] HE D, LIU G, YANG J, et al.Specific high-sensitivity enzymatic molecular detection system termed RPA-based CRISPR-Cas13a for Duck Tembusu virus diagnostics[J].Bioconjugate Chemistry, 2022, 33(6):1232-1240.
[23] PEROUTKA III R J, ORCUTT S J, STRICKLER J E, et al.SUMO fusion technology for enhanced protein expression and purification in prokaryotes and eukaryotes[J].Methods in Molecular Biology, 2011, 705:15-30.
[24] KIM D S, KIM S W, SONG J M, et al.A new prokaryotic expression vector for the expression of antimicrobial peptide abaecin using SUMO fusion tag[J].BMC Biotechnology, 2019, 19(1):13.
[25] PARK A R, KIM S W, KIM S Y, et al.Expression of antimicrobial peptide (AMP), cecropin B, in a fused form to SUMO tag with or without three-glycine linker in Escherichia coli and evaluation of bacteriolytic activity of the purified AMP[J].Probiotics Antimicrob Proteins, 2021, 13(6):1780-1789.
[26] TAN M S, TEH Y H, HO K L, et al.An application of pET SUMO protein expression system in Escherichia coli:Cloning, expression, purification, and characterisation of native kras4B (G12V) oncoprotein[J].Protein Journal, 2020, 39(1):54-61.
[27] GOOTENBERG J S, ABUDAYYEH O O, LEE J W, et al.Nucleic acid detection with CRISPR-Cas13a/C2c2[J].Science, 2017, 356(6336):438-442.
[28] NALEFSKI E A, PATEL N, LEUNG P J Y, et al.Kinetic analysis of Cas12a and Cas13a RNA-guided nucleases for development of improved CRISPR-based diagnostics[J].iScience, 2021, 24(9):102996.
[29] IKEDA S, KUBOTA T, YUKI M, et al.Hybridization-sensitive fluorescent DNA probe with self-avoidance ability[J].Organic & Biomolecular Chemistry, 2010, 8(3):546-551.

Expression, Purification and Activity Analysis of LwaCas13a Protein

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