Recombinant Expression and Detection of Antimicrobial Activity of Cec4a

doi:10.13523/j.cb.2105046

China Biotechnology

2021, Vol. 41

Issue (10): 12-18 DOI: 10.13523/j.cb.2105046

Recombinant Expression and Detection of Antimicrobial Activity of Cec4a

CHEN Su-fang¹,XIA Ming-yin¹,ZENG Li-yan¹,AN Xiao-qin¹,TIAN Min-fang¹,PENG Jian^1,^2,^**(

)

1 School of Biology and Medical Engineering/Basic Medical College,Guizhou Medical University,Guiyang 550025,China
2 Key Laboratory of Environmental Pollution and Disease Control of Ministry of Education, Guiyang 550025,China

Download:

HTML

PDF(992KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective: To construct the recombinant expression system of Cec4a, and to obtain the recombinant protein by induced expression and detect the antibacterial activity of the product. Methods: Based on the primers designed according to the sequence of Cec4a, the mature peptide part of Cec4a gene was amplified by PCR. Recombinant prokaryotic expression plasmid was constructed using prokaryotic expression vector (pCold-SUMO) and transformed into E. coli C41 (DE3) competent cells, which were induced by IPTG. His-SUMO labeled recombinant Cec4a fusion protein was purified by Ni-NTA affinity chromatography. The target protein was purified by Ni-NTA affinity chromatography after SUMO protease digestion. Acinetobacter baumannii (ATCC19606) was used to detect the antibacterial activity of the product. Results: pCold-SUMO-Cec4a prokaryotic expression plasmid was successfully constructed, and the sequencing analysis was consistent with the expected results. The expression level of Cec4a fusion protein was 42.8mg/L, and the MIC of purified Cec4a recombinant protein against Acinetobacter baumannii was 4 μg/mL. Conclusion: The recombinant Cec4a protein with antibacterial activity was successfully constructed and purified by Ni-NTA affinity chromatography. It lays a foundation for further study on the biological activity, the relationship between the structure and function of Cec4a.

Key words： Cec4a SUMO Acinetobacter baumannii Recombinant expression

Received: 25 May 2021 Published: 08 November 2021

ZTFLH:

Q819

Corresponding Authors: Jian PENG E-mail: pjf66666@126.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Su-fang CHEN
	Ming-yin XIA
	Li-yan ZENG
	Xiao-qin AN
	Min-fang TIAN
	Jian PENG

Cite this article:

CHEN Su-fang,XIA Ming-yin,ZENG Li-yan,AN Xiao-qin,TIAN Min-fang,PENG Jian. Recombinant Expression and Detection of Antimicrobial Activity of Cec4a. China Biotechnology, 2021, 41(10): 12-18.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2105046 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I10/12

Table 1 Codon optimization

Fig.1 Codon optimization of the Cec4a fragment

Fig.2 PCR amplification of the Cec4a fragment

Fig.3 SDS-PAGE analysis of the fusion protein MW: Molecular weight marker; Ø: Non-induced bacterial culture (negative control); 16, 30 and 37 are different temperatures(℃)

Fig.4 SDS-PAGE analysis of purified fusion proteins MW: Protein molecular weight marker. IN: Supernatant. FT: Passing through liquid. W: Miscellaneous protein. E: Objective Protein

Fig.5 Target protease digestion MW: Protein marker. IN: Input. FT: Passing through liquid. A11, A12: SUMO-Cec4a protein component. B9, B10: Elution. C3-C7: SUMO protease and target protein Cec4a

Fig.6 Purification of target protein MW: Protein marker. IN: Enzyme cut mixture. A10: Elution. D-E: SUMO protease. E8-E12: Target protein Cec4a

Table 2 Effects of salts, and serum on peptide activity against A. baumannii, MIC (μg/mL)

Fig.7 Transmission electron microscopy showed that the antimicrobial peptide Cec4a could lead to the cleavage of A. baumannii in 1~3 h (a) Bacteria untreated with antimicrobial peptide (b-d) Morphological changes of bacteria after treatment with antimicrobial peptide Cec4a for 1~3 h


[1]	Fang S L, Wang L, Fang Q, et al. Characterization and functional study of a Cecropin-like peptide from the Chinese oak silkworm, Antheraea pernyi. Archives of Insect Biochemistry and Physiology, 2017, 94(1): e21368. doi: 10.1002/arch.v94.1

[2]	Zhou J, Fang N N, Zheng Y, et al. Identification and characterization of two novel C-type lectins from the larvae of housefly, Musca domestica L. Archives of Insect Biochemistry and Physiology, 2018, 98(3): e21467. doi: 10.1002/arch.v98.3

[3]	Lee C R, Lee J H, Park M, et al. Biology of Acinetobacter baumannii: pathogenesis, antibiotic resistance mechanisms, and prospective treatment options. Frontiers in Cellular and Infection Microbiology, 2017, 7: 55.

[4]	Eraç B, Yilmaz F F, Hosgör Limoncu M, et al. Investigation of the virulence factors of multidrug-resistant Acinetobacter baumannii isolates. Mikrobiyoloji Bulteni, 2014, 48(1): 70-81. doi: 10.5578/mb.6981

[5]	彭建, 赵行行, 吴兆颖, 等. 抗菌肽Cec4的结构改造及抗菌活性研究. 生物技术, 2019, 29(4): 330-335.

[5]	Peng J, Zhao X X, Wu Z Y, et al. Structural modification and antibacterial related activity study of antimicrobial peptide Cec4. Biotechnology, 2019, 29(4): 330-335.

[6]	Ptaszyńska N, Olkiewicz K, Okońska J, et al. Peptide conjugates of lactoferricin analogues and antimicrobials-design, chemical synthesis, and evaluation of antimicrobial activity and mammalian cytotoxicity. Peptides, 2019, 117: 170079. doi: S0196-9781(19)30037-3 pmid: 30959143

[7]	Panavas T, Sanders C, Butt T R. SUMO fusion technology for enhanced protein production in prokaryotic and eukaryotic expression systems. Methods in Molecular Biology (Clifton, N J), 2009, 497: 303-317.

[8]	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. BMC Biotechnology, 2019, 19(1): 13. doi: 10.1186/s12896-019-0506-x

[9]	刘杞, 石小枫, 罗娅, 等. 重组人肝再生增强因子原核表达载体的构建及其在大肠杆菌中的表达. 中华肝脏病杂志, 2000, 8(1): 9-11. pmid: 10712774

[9]	Liu Q, Shi X F, Luo Y, et al. Construction of prokaryotic expression vector of hALR and its expression in E.coli. Chinese Journal of Hepatology, 2000, 8(1): 9-11. pmid: 10712774

[10]	Rajan R, Weisshaar J C. Insights into the effects of antimicrobial peptides on live E. coli cells using time-lapse fluorescence microscopy. Biophysical Journal, 2015, 108(2): 548a.

[11]	Kaur K, Park H, Pandey N, et al. Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy. Journal of Biological Chemistry, 2017, 292(24): 10230-10238. doi: 10.1074/jbc.M117.789982

[12]	Cuijpers S A G, Willemstein E, Vertegaal A C O. Converging small ubiquitin-like modifier (SUMO) and ubiquitin signaling: improved methodology identifies co-modified target proteins. Molecular & Cellular Proteomics, 2017, 16(12): 2281-2295. doi: 10.1074/mcp.TIR117.000152

[13]	Lao M X, Zhan Z P, Li N, et al. Role of small ubiquitin-like modifier proteins-1 (SUMO-1) in regulating migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis. Experimental Cell Research, 2019, 375(1): 52-61. doi: 10.1016/j.yexcr.2018.12.011

[14]	Wang Z F, Zhu W G, Xu X Z. Ubiquitin-like modifications in the DNA damage response. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2017, 803-805: 56-75. doi: 10.1016/j.mrfmmm.2017.07.001

[15]	Oravcová M, Boddy M N. Recruitment, loading, and activation of the Smc5-Smc6 SUMO ligase. Current Genetics, 2019, 65(3): 669-676. doi: 10.1007/s00294-018-0922-9 pmid: 30600397

[16]	Lamberti A, Sanges C, Longo O, et al. Analysis of nickel-binding peptides in a human hepidermoid cancer cell line by Ni-NTA affinity chromatography and mass spectrometry. Protein and Peptide Letters, 2008, 15(10): 1126-1131. doi: 10.2174/092986608786071157

[17]	Moser A C, White B, Kovacs F A. Measuring binding constants of his-tagged proteins using affinity chromatography and Ni-NTA-immobilized enzymes. Methods in Molecular Biology (Clifton, N J), 2014, 1129: 423-434.

[18]	Hara N, Futo S, Sekiguchi S, et al. A new method to obtain high DNA transformation efficiency of E.coli competent cells. Nucleic Acids Research, 1988, 16(17): 8727. pmid: 3047689

[19]	Yu D D, Wang Y, Zhang S Q, et al. An ultrasensitive stain for negative protein detection in SDS-PAGE via 4', 5'-dibromofluorescein. Journal of Proteomics, 2017, 165: 21-25. doi: 10.1016/j.jprot.2017.06.014

[20]	Kinoshita E, Kinoshita-Kikuta E, Koike T. Zn(II)-Phos-tag SDS-PAGE for separation and detection of a DNA damage-related signaling large phosphoprotein. Methods in Molecular Biology (Clifton, N J), 2017, 1599: 113-126.

[21]	张雪洋, 赵华, 赵红宇, 等. 人釉原蛋白基因在大肠杆菌中的融合表达. 华西口腔医学杂志, 2008, 26(1): 27-30.

[21]	Zhang X Y, Zhao H, Zhao H Y, et al. Expression and purification of human amelogenin in Escherichia coli. West China Journal of Stomatology, 2008, 26(1): 27-30.

[22]	姚玲玲, 王家宁, 黄永章, 等. pET15b-PEP-1-CAT原核表达质粒的构建及PEP-1-CAT融合蛋白的表达与纯化. 南方医科大学学报, 2006, 26(9): 1319-1325.

[22]	Yao L L, Wang J N, Huang Y Z, et al. Construction of prokaryotic expression plasmid pET15b-PEP-1-CAT and expression and purification of PEP-1-CAT fusion protein. Journal of Southern Medical University, 2006, 26(9): 1319-1325.

[23]	史鹏伟, 高艳彬, 卢志阳, 等. 抗菌肽LL-37对鲍曼不动杆菌生物膜的抑制作用. 南方医科大学学报, 2014, 34(3): 426-429.

[23]	Shi P W, Gao Y B, Lu Z Y, et al. Effect of antibacterial peptide LL-37 on the integrity of Acinetobacter baumannii biofilm. Journal of Southern Medical University, 2014, 34(3): 426-429.

[24]	Molchanova N, Hansen P, Franzyk H. Advances in development of antimicrobial peptidomimetics as potential drugs. Molecules, 2017, 22(9): 1430. doi: 10.3390/molecules22091430

[25]	Chionis K, Krikorian D, Koukkou A I, et al. Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin. Journal of Peptide Science, 2016, 22(11-12): 731-736. doi: 10.1002/psc.2939 pmid: 27862650

[26]	张辉, 段招军, 朱建高, 等. 人新型干扰素κ的基因克隆、表达、纯化及其抗病毒活性的初步研究. 中华实验和临床病毒学杂志, 2005, 19(3): 223-226. pmid: 16261202

[26]	Zhang H, Duan Z J, Zhu J G, et al. Cloning, expression and purification of interferon-kappa, a novel human interferon, and its antiviral activity. Chinese Journal of Experimental and Clinical Virology, 2005, 19(3): 223-226. pmid: 16261202

[27]	Peleg Y, Unger T. Resolving bottlenecks for recombinant protein expression in E. coli. Methods in Molecular Biology (Clifton, N J), 2012, 800: 173-186.

[28]	Gileadi O. Recombinant protein expression in E. coli: a historical perspective. Methods in Molecular Biology (Clifton, N J), 2017, 1586: 3-10.

[29]	赵少若, 王孟月, 白晶晶, 等. 非洲猪瘟病毒DP96R蛋白的表达与单克隆抗体的制备. 畜牧与兽医, 2021, 53(2): 77-81.

[29]	Zhao S R, Wang M Y, Bai J J, et al. Expression and monoclonal antibody preparation of the DP96R recombinant protein of African swine fever virus. Animal Husbandry & Veterinary Medicine, 2021, 53(2): 77-81.

[1]	LE Yi-lin,FU Yu,NI Li,SUN Jian-zhong. *Expression and Characterization of a Thermostable Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophile Thermotoga neapolitana* and Its Application in Acetyl-CoA Production**[J]. China Biotechnology, 2020, 40(3): 72-78.

[2]	XUE Rui,YAO Lin,WANG Rui,LUO Zheng-shan,XU Hong,LI Sha. Advances and Applications of Recombinant Mussel Foot Proteins[J]. China Biotechnology, 2020, 40(11): 82-89.

[3]	HAN Ting-han,ONG Xue-mei,ING Ya-fang,U Chen,ZHANG Kun-xiao,AO song,U Heng-hao. Cloning, Expression and Characterization of a Heat-Labile Uracil-DNA lycosylase from Scophthalmus maximus[J]. China Biotechnology, 2019, 39(10): 34-43.

[4]	Shi-jie LI,Yan-kun YANG,Meng LIU,Zhong-hu BAI,Jian JIN. Efficient Expression of SUMO Protease Ulp1 and Used to Express and Purified scFv by His-SUMO tag[J]. China Biotechnology, 2018, 38(3): 51-61.

[5]	ZENG Jie. Development and Application of L-Asparaginase with Better Performance and Advances in Recombinant Expression[J]. China Biotechnology, 2017, 37(11): 123-131.

[6]	RAO Jing-jing, JING Yi-xian, ZOU Ming-yue, HU Xiao-lei, LIAO Fei, YANG Xiao-lan. Clone, Expression and Characterization of the Uricase from Meyerozyma guilliermondii[J]. China Biotechnology, 2017, 37(11): 74-82.

[7]	ZHAO Yi-jin, WANG Teng-fei, WANG Jun-qing, WANG Rui-ming. *Surface Display of Tres Using CotC as a Molecular Vector on Bacillus subtilis* Spores**[J]. China Biotechnology, 2017, 37(1): 71-80.

[8]	LI Meng-yue, WANG Teng-fei, WANG Jun-qing, ZHAO Yi-jin, CHENG Cheng, WANG Rui-ming. Expression of Trehalose Synthase Gene in Pichia pastoris[J]. China Biotechnology, 2016, 36(2): 73-80.

[9]	DING Yi, WU Hai-ying, SHI Ji-ping, SUN Jun-song. Current Progress in Recombinant Systems for Expression of Hydrogenases[J]. China Biotechnology, 2015, 35(5): 109-118.

[10]	LI Cui-lin, ZHANG Fan, CHEN Dan-yang, WANG Hao, GUO Qiang, DU Jun. *Study of Prokaryotic Expression and Biological Activity of Homo sapiens* Kras Protein**[J]. China Biotechnology, 2014, 34(8): 1-6.

[11]	YANG Bo, CHEN Hai-qin, SONG Yuan-da, ZHANG Hao, CHEN Wei. Study of the Enzymatic Function of Myosin Cross Reactive Antigen from Bifidobacterium animalis[J]. China Biotechnology, 2012, 32(12): 30-36.

[12]	CHEN Xiao-jing, CHEN Xiao-mei, WANG Yang, SHI Hui-li, HUO Ke-ke. Prokaryotic Expression and Purification of Human SCYL1-BP1 and Its Identification[J]. China Biotechnology, 2012, 32(09): 1-8.

[13]	GAO Bing-miao, LI Bao-zhu, WU Yong, LIN Bo, ZHU Xiao-peng, ZHANGSUN Dong-ting, LUO Su-lan. Expression, Purification and Identfication of Recombinant Conotoxin GeXIVAWT[J]. China Biotechnology, 2012, 32(09): 34-40.

[14]	WEI Hai-Chao, ZHANG Yan, FAN Yao-Chun, LI Chuan-Yi, WEN Yu-Ling, CHEN Yuan-Ding. Expression of NSP1 of a Group A Human Rotavirus and Immunological Properties[J]. China Biotechnology, 2010, 30(03): 15-21.

[15]	ZHANG Hai-Miao, LIU Xiao-Ju, TIAN Hai-Shan, MO Xiao-Shan, DENG Lin, LI Jiao-Kun. Expression and Purification of Fusion Protein with SUMO of Human Epidermal Growth Factor[J]. China Biotechnology, 2010, 30(03): 74-78.

Viewed

Full text

Abstract

Cited

Shared

Discussed