利用GAP启动子在毕赤酵母中组成型表达人鹅型溶菌酶2 <sup>*</sup>

doi:10.13523/j.cb.20181007

中国生物工程杂志

2018, Vol. 38

Issue (10): 55-63 DOI: 10.13523/j.cb.20181007

技术与方法

利用GAP启动子在毕赤酵母中组成型表达人鹅型溶菌酶2 ^*

黄鹏^1,^**,***(

),阎丽萍^2,^**,张宁³,石金磊⁴

1 上海健康医学院临床医学院上海 201318
2 青岛大学附属中心医院青岛 266042
3 上海健康医学院基础医学院上海 201318
4 上海科技大学生命科学与技术学院上海 201210

Constitutive Expression of Human Goose-type Lysozyme 2 in Pichia pastoris Using the GAP Promoter

Peng HUANG^1,^**,***(

),Li-ping YAN^2,^**,Ning ZHANG³,Jin-lei SHI⁴

1 College of Clinical Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
2 Clinical Laboratory, Affiliated Central Hospital of Qingdao University, Qingdao 266042, China
3 College of Basic Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
4 School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China

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摘要：

利用甘油醛三磷酸脱氢酶(glyceraldehydes-3-phosphatedehydrogenase,GAP)启动子在毕赤酵母中表达人鹅型溶菌酶2(human goose-type lysozyme 2,hLysG2),并在小试规模建立一套有效的重组hLysG2(recombinant hLysG2,rhLysG2)生产工艺流程。根据毕赤酵母密码子偏爱性设计并人工合成hLysG2基因,将其连接至pGAPZαA 质粒中,构建重组表达质粒pGAPZαA-hLysG2。将重组表达载体线性化后电转化毕赤酵母GS115感受态细胞,通过Zeocin抗性筛选获取高拷贝重组菌株,并在5L生物反应器中进行发酵培养。发酵60h后发酵液上清酶活性达到最高, 发酵液上清经SDS-PAGE及Western blot检测证实rhLysG2得到表达。与诱导型表达相比,组成型表达发酵时间缩短了48h,上清中rhLysG2总活性提高了23.8%;使用甲壳素亲和层析和分子筛层析对rhLysG2进行纯化后,每升发酵液上清可纯化到187.4mg重组蛋白,纯化产物纯度达99.0%以上;浊度测定法分析显示,在pH 5.6、30℃和0.1mol/L Na ⁺的条件下,rhLysG2可达到最大酶活性13 500U/mg。利用GAP启动子在毕赤酵母中成功表达了高纯度和高活性的rhLysG2,避免了甲醇的使用,缩短了发酵时间,提高了蛋白产量,为将rhLysG2开发为新型抗耐药菌药物奠定了基础。

关键词： 人鹅型溶菌酶2; 毕赤酵母; GAP启动子; 组成型表达; 杀菌活性

Abstract:

This study aimed to achieve the constitutive expression of human goose-type lysozyme 2 (hLysG2) in Pichia pastoris using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and to establish an efficient strategy for the production of recombinant hLysG2 (rhLysG2) on a bench scale. The hLysG2 gene was synthesized according to the codon usage preference of P. pastoris and cloned into pGAPZαA vector. The resulting pGAPZαA-hLysG2 plasmid was linearized and transformed into competent P. pastoris GS115, followed by Geneticin screening. The transformants with higher Geneticin resistance were selected to investigate the constitutive expression of rhLysG2 in P. pastoris. The lytic activity of rhLysG2 in the fermentation broth reached its peak after 60h of cultivation. SDS-PAGE and Western blot analysis showed that rhLysG2 was successfully secreted into the fermentation broth. There were a 23.8% increase in the total lytic activity and a 48h reduction in the cultivation time in comparison with those of the P. pastoris strain integrated with the pPIC9K-hLysG2 plasmid. Using chitin affinity and size-exclusion chromatography, rhLysG2 was purified with a yield of 187.4mg/L of fermentation supernatant, above 99.9% purity and a specific activity of 13 500U/mg under the condition of pH 5.6, 0.1mol/L of Na ⁺, 30℃. In conclusion, rhLysG2 was expressed at high level in P. pastoris by codon optimization and had in vitro bactericidal activity against some pathogenic bacteria, which has laid a solid foundation for its possible future pharmaceutical applications.

Key words: Human goose-type lysozyme 2 Pichia pastoris GAP promoter Constitutive expression Bactericidal activity

收稿日期: 2018-04-16 出版日期: 2018-11-09

ZTFLH:

R392-33R392.11

基金资助: * 上海市卫生计生委科研课题(201740161);上海市自然科学基金(15ZR1421800)

通讯作者: 黄鹏,阎丽萍 E-mail: huangp_15@sumhs.edu.cn

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引用本文:

黄鹏,阎丽萍,张宁,石金磊. 利用GAP启动子在毕赤酵母中组成型表达人鹅型溶菌酶2 ^*[J]. 中国生物工程杂志, 2018, 38(10): 55-63.

Peng HUANG,Li-ping YAN,Ning ZHANG,Jin-lei SHI. Constitutive Expression of Human Goose-type Lysozyme 2 in Pichia pastoris Using the GAP Promoter. China Biotechnology, 2018, 38(10): 55-63.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181007 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I10/55

表1 本研究所用引物

图1 目的基因扩增及重组质粒酶切鉴定

图2 目的基因扩增及重组质粒酶切鉴定

图3 阳性重组毕赤酵母菌株PCR鉴定

图4 发酵液上清活性增长曲线

图5 发酵液上清的SDS-PAGE

图6 甲壳素亲和层析洗脱曲线

图7 rhLysG2纯化蛋白SDS-PAGE (上)与Western blot分析 (下)

图8 纯化rhLysG2的HPLC分析

表2 rhLysG2纯化过程收率分析

图9 pH、离子浓度和温度对rhLysG2活性和稳定性的影响

表3 金属离子对rhLysG2活性的影响

图10 rhLysG2杀菌活性分析(n=3)

[1]	Irwin D M . Evolution of the vertebrate goose-type lysozyme gene family. BMC Evolutionary Biology, 2014,14(1):188-202. doi: 10.1186/s12862-014-0188-x pmid: 4243810
[2]	Weaver L H, Grutter M G, Remington S J , et al. Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution. Journal of Molecular Evolution, 1985,21(2):97-111. doi: 10.1007/BF02100084 pmid: 6442995
[3]	Prager E M . Adaptive evolution of lysozyme: changes in amino acid sequence, regulation of expression and gene number. EXS, 1996,75:323-345. doi: 10.1007/978-3-0348-9225-4_17 pmid: 8765307
[4]	Canfield R E, Mcmurry S . Purification and characterization of a lysozyme from goose egg white. Biochemical and Biophysical Research Communications, 1967,26(1):38-42. doi: 10.1016/0006-291X(67)90249-5 pmid: 6030253
[5]	Ko J, Wan Q, Bathige S D , et al. Molecular characterization, transcriptional profiling, and antibacterial potential of G-type lysozyme from seahorse (Hippocampus abdominalis). Fish & Shellfish Immunology, 2016,58:622-630. doi: 10.1016/j.fsi.2016.10.014 pmid: 27732899
[6]	Liu Q N, Xin Z Z, Zhang D Z , et al. Molecular identification and expression analysis of a goose-type lysozyme (LysG) gene in yellow catfish Pelteobagrus fulvidraco. Fish & Shellfish Immunology, 2016,58:423-428. doi: 10.1016/j.fsi.2016.09.034 pmid: 27645907
[7]	Gao C, Fu Q, Zhou S , et al. The mucosal expression signatures of g-type lysozyme in turbot (Scophthalmus maximus) following bacterial challenge. Fish & Shellfish Immunology, 2016,54:612-619. doi: 10.1016/j.fsi.2016.05.015 pmid: 27189917
[8]	Wei S, Huang Y, Huang X , et al. Molecular cloning and characterization of a new G-type lysozyme gene (Ec-lysG) in orange-spotted grouper, Epinephelus coioides. Developmental and Comparative Immunology, 2014,46(2):401-412. doi: 10.1016/j.dci.2014.05.006
[9]	Buonocore F, Randelli E, Trisolino P , et al. Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L.). Molecular Immunology, 2014,62(1):10-18.
[10]	Wang R, Feng J, Li C , et al. Four lysozymes (one c-type and three g-type) in catfish are drastically but differentially induced after bacterial infection. Fish & Shellfish Immunology, 2013,35(1):136-145. doi: 10.1016/j.fsi.2013.04.014 pmid: 23639933
[11]	Guo Y, He H . Identification and characterization of a goose-type lysozyme from sewage snail Physa acuta. Fish & Shellfish Immunology, 2014,39(2):321-325. doi: 10.1016/j.fsi.2014.05.029 pmid: 24882016
[12]	Huang P, Li W S, Xie J , et al. Characterization and expression of hLysG2, a basic goose- type lysozyme from the human eye and testis. Molecular Immunology, 2011,48(4):524-531. doi: 10.1016/j.molimm.2010.10.008 pmid: 21093056
[13]	Irwin D M, Gong Z . Molecular evolution of vertebrate goose-type lysozyme genes. Journal of Molecular Evolution, 2003,56(2):234-242. doi: 10.1007/s00239-002-2396-z
[14]	黄鹏, 杨智昉, 谢君 , 等. 人g2型溶菌酶的多克隆抗体制备及其在睾丸中的表达分析. 免疫学杂志, 2016,32(10):842-847.
	Huang P, Yang Z F, Xie J , et al. Preparation of polyclonal antibody of human goose-type lysozyme 2 and its expression in human testis. Immunology Journal, 2016,32(10):842-847.
[15]	黄鹏, 杨智昉, 鲍建瑛 , 等. 人精浆中g2型溶菌酶的纯化及其酶学活性分析. 细胞与分子免疫学杂志, 2017,33(3):320-325.
	Huang P, Yang Z F, Bao J Y , et al. Purification of human goose-type lysozyme 2 (hLysG2) from human seminal plasma and analysis of its enzymatic properties. Chinese Journal of Cellular and Molecular Immunology, 2017,33(3):320-325.
[16]	Stahlmann R . Antibiotics for treatment of infections by methicillin-resistant Staphylococcus aureus (MRSA). Pneumologie, 2014,68(10):676-684. doi: 10.1055/s-00000059
[17]	Holmes N E, Howden B P . What’s new in the treatment of serious MRSA infection? Current Opinion in Infectious Diseases, 2014,27(6):471-478. doi: 10.1097/QCO.0000000000000101 pmid: 25211361
[18]	Daly R, Hearn M T . Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. Journal of Molecular Recognition, 2005,18(2):119-138. doi: 10.1109/4.953490 pmid: 15565717
[19]	Macauley-Patrick S, Fazenda M L, Mcneil B , et al. Heterologous protein production using the Pichia pastoris expression system. Yeast, 2005,22(4):249-270. doi: 10.1002/yea.1208 pmid: 15704221
[20]	Jahic M, Veide A, Charoenrat T , et al. Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnology Progress, 2006,22(6):1465-1473. doi: 10.1002/btpr.v22:6
[21]	Ahmad M, Hirz M, Pichler H , et al. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied Microbiology and Biotechnology, 2014,98(12):5301-5317. doi: 10.1007/s00253-014-5732-5 pmid: 4047484
[22]	Romanose M A, Scorer C A, Clare J J . Foreign gene expression in yeast: a review. Yeast, 1992,8(6):423-488. doi: 10.1002/yea.320080602 pmid: 1502852
[23]	Waterham H R, Digan M E, Koutz P J , et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene, 1997,186(1):37-44. doi: 10.1016/S0378-1119(96)00675-0 pmid: 9047342
[24]	Zhang A L, Luo J X, Zhang T Y , et al. Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter. Acta Genetica Sinica, 2004,31(6):552-557. doi: 10.1007/BF02873086 pmid: 15490871
[25]	Menéndez C, Martínez D, Trujillo L E , et al. Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris. Applied Microbiology and Biotechnology, 2013,97(3):1201-1212. doi: 10.1007/s00253-012-4270-2
[26]	钱凯, 张晶晶, 吴素平 , 等. 利用GAP 启动子在毕赤酵母中表达与纯化GLP-1类似物. 中国生物工程杂志, 2015,35(5):66-73. doi: 10.13523/j.cb.20150510
	Qian K, Zhang J J, Wu S P , et al. Constituted expression and purification of glucagon-like peptide-1 analogue in Pichia pastoris using GAP promoter. China Biotechnology, 2015,35(5):66-73. doi: 10.13523/j.cb.20150510
[27]	黄鹏, 李文姝, 谢君 , 等. 甲壳素亲和层析在人g2型溶菌酶制备中的应用. 免疫学杂志, 2015,31(5):430-435.
	Huang P, Li W S, Xie J , et al. Application of chitin affinity chromatography in purification of recombinant hLysG2. Immunology Journal, 2015,31(5):430-435.
[28]	Klockars M, Reitamo S . Tissue distribution of lysozyme in man. Journal of Histochemistry & Cytochemistry, 1975,23(12):932-940.
[29]	Mason D Y, Taylor C R . The distribution of muramidase (lysozyme) in human tissues. Journal of Clinical Pathology, 1975,28(2):124-132. doi: 10.1136/jcp.28.2.124 pmid: 475611

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