Preparation of <i>β</i>-Casein Based on <i>Escherichia coli</i> Expression System

AI Zhengwen

doi:10.13386/j.issn1002-0306.2022090232

Volume 44 Issue 16

Aug. 2023

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Science and Technology of Food Industry > 2023 > 44(16): 131-138. > DOI: 10.13386/j.issn1002-0306.2022090232

AI Zhengwen. Preparation of β-Casein Based on Escherichia coli Expression System[J]. Science and Technology of Food Industry, 2023, 44(16): 131−138. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022090232.

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Preparation of β-Casein Based on Escherichia coli Expression System

AI Zhengwen

State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Bright Dairy & Food Co., Ltd., Dairy Research Institute, Shanghai 200436, China

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Received Date: September 21, 2022
Available Online: June 19, 2023

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Abstract

Abstract

The β-casein of bovine milk contains a variety of variants, among which A1-β-casein (A1) and A2-β-casein (A2) are the two most common variants. Because of only few differences between with A1 and A2 in amino acid sequence, it is difficult to preparation of A1 and A2 proteins with higher purity by conventional separation and purification methods. In this study, recombinant plasmid of pET28a（+）-CSN2-A1 and pET28a （+）-CSN2-A2, which contained the target genes of CSN2-A1 and CSN2-A2 were constructed by molecular biological methods, respectively. Then two recombinant vectors were introduced into Escherichia coli BL21 for induced expression and purification, respectively. The results showed that abundant of proteins could be obtained by induced expression at 0.2 mmol/L IPTG at 37 ℃ for 4 h. However, the results of SDS-PAGE showed that the target proteins were expressed in the form of inclusion bodies, and existed in the pellet after cell disruption. In addition, the purity of more than 90% (SDS-PAGE) of the A1 and A2 recombinant proteins were obtained by several processes, which contained dissolution, nickel affinity chromatography, renaturation and qualification. Thereby, it could provide a new way for the preparation of A1 and A2 proteins.
- β-casein,
- Escherichia coli,
- prokaryotic expression,
- CSN2-A1,
- CSN2-A2

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[1]	李建斌, 刘文娇, 王秀革, 等. 奶牛β-酪蛋白基因分型及A2型牛群培育技术研究进展[J]. 中国奶牛,2017(6):31−33. [LI Jiangbin, LIU wenjiao, WANG Xiuge, et al. Research progress on β-casein genotyping of dairy cattle and breeding technology of A2 herds[J]. China Dairy Cattle,2017(6):31−33. LI Jiangbin, LIU wenjiao, WANG Xiuge, et al. Research progress on β-casein genotyping of dairy cattle and breeding technology of A2 herds[J]. China Dairy Cattle, 2017(6): 31-33.
[2]	冯慿, 丁晓静, 高铁, 等. 毛细管区带电泳测定液态奶及奶粉中的A2β-酪蛋白及总β-酪蛋白[J]. 色谱,2020,38(6):722−729. [FENG Ping, DING Xiaojing, GAO Tie, DU Ruyun, et al. Quantification of A2 variant and total β-casein content in cow milk based liquid and powder products by capillary zone electrophoresis[J]. Chinese Journal of Chromatography,2020,38(6):722−729. FENG Ping, DING Xiaojing, GAO Tie, DU Ruyun, et al. Quantification of A2 variant and total β-casein content in cow milk based liquid and powder products by capillary zone electrophoresis[J]. Chinese Journal of Chromatography, 2020, 38(6): 722-729.
[3]	王丹, 王青云, 王慧敏, 等. 牛乳β-酪蛋白遗传多态性及A2型乳制品研究进展[J]. 中国奶牛,2021(3):40−43. [WANG Dan, WANG Qingyun, WANG Huimin, et al. Research progress on genetic polymorphism of β-casein in bovine milk and type A2 dairy products[J]. China Dairy Cattle,2021(3):40−43. WANG Dan, WANG Qingyun, WANG Huimin, et al. Research progress on genetic polymorphism of β-casein in bovine milk and type A2 dairy products[J]. China Dairy Cattle, 2021(3): 40-43.
[4]	CAROLI A M, SAVINO S, BULGARI O, et al. Detecting β-casein variation in bovine milk[J]. Molecules,2016,21(2):141−147. doi: 10.3390/molecules21020141
[5]	陈龙, 付王艳, 方琼燕, 等. 牛乳中β-酪蛋白基因分型及β-酪啡肽-7的研究进展[J]. 中国乳品工业,2019,47(08):29−34. [CHEN Long, FU Wangyan, FANG Qiongyan, et al. Recent advances of β-casein genotyping in bovine milk and potential impact of β-casomorphin-7 on human health[J]. China Dairy Industry,2019,47(08):29−34. CHEN Long, FU Wangyan, FANG Qiongyan, et al. Recent advances of β-casein genotyping in bovine milk and potential impact of β-casomorphin-7 on human health[J]. China Dairy Industry, 2019, 47(08): 29-34.
[6]	罗卉卉, 金素钰, 黄林, 等. 牦牛β-酪蛋白A1、A2型遗传变异体的基因和蛋白质水平分析[J]. 黑龙江畜牧兽医,2020(06):133−135,140,158. [LUO Benben, JIN Suyu, HUANG Lin, et al. Analysis of yak β-casein A1 and A2 genetic variants on the level of gene and protein[J]. Heilongjiang Animal Science and Veterinary Medicine,2020(06):133−135,140,158. LUO Benben, JIN Suyu, HUANG Lin, et al. Analysis of yak β-casein A1 and A2 genetic variants on the level of gene and protein[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020,（06）: 133-135, 140, 158.
[7]	SUN Jianqin, XU Leiming, XIA Lu, et al. Effects of milk containing only A2 beta casein versus milk containing both A1 and A2 beta casein proteins on gastrointestinal physiology, symptoms of discomfort, and cognitive behavior of people with self-reported intolerance to traditional cows' milk[J]. Nutrition Journal,2016,15(1):35−50.
[8]	DANILOSKI D, CUNHA N, MCCARTHY N A, et al. Health-related outcomes of genetic polymorphism of bovine β-casein variants: A systematic review of randomised controlled trials[J]. Trends in Food Science & Technology,2021,111(6):233−248.
[9]	刘泽阳, 李明, 吴佩泽, 等. 液相色谱-高分辨串联质谱法检测牛奶中A1和A2 β-酪蛋白[J]. 吉林大学学报(理学版),2021,59(3):696−702. [LIU Zeyang, LI Ming, WU Peize, et al. Deterination of A1 and A2 β-casein in milk by liquid chromatography-high resolution tandem mass spectrometry[J]. Journal of Jilin University (Science Edition),2021,59(3):696−702. LIU Zeyang, LI Ming, WU Peize, et al. Deterination of A1 and A2 β-casein in milk by liquid chromatography-high resolution tandem mass spectrometry[J]. Journal of Jilin University(Science Edition), 2021, 59(3): 696-702.
[10]	常硕. 牛奶中A1和A2 β-酪蛋白的检测与分析[J]. 中国奶牛,2018(3):48−50. [CHANG Shuo. Detection and analysis of A1 β-casein and A2 β-casein in bovine milk[J]. China Dairy Cattle,2018(3):48−50. CHANG Shuo. Detection and analysis of A1 β-casein and A2 β-casein in bovine milk[J]. China Dairy Cattle, 2018(3): 48-50.
[11]	MAKINO T, SKRETAS G, GEORGIOU G. Strain engineering for improved expression of recombinant proteins in bacteria[J]. Microbial Cell Factories,2011,10(1):32−41. doi: 10.1186/1475-2859-10-32
[12]	HEMAMALINI N, EZHILMATHi S, MERCY A A. Recombinant protein expression optimization in Escherichia coli: A review[J]. Indian Journal of Animal Research,2020,54(6):653−660.
[13]	GOYAL V, KOHLI I, AMBASTHA V, et al. Synthetic biology tools: Engineering microbes for biotechnological applications[M]. New and Future Developments in Microbial Biotechnology and Bioengineering, 2022: 369−98.
[14]	周正富, 庞雨, 张维, 等. 乳蛋白重组表达与人造奶生物合成: 全球专利分析与技术发展趋势[J]. 合成生物学,2021,2(5):764−777. [ZHOU Zhengfu, PANG Yu, ZHANG Wei, et al. Recombinant expression of milk proteins and biosynthesis of animal-free milk: analysis on related patents and trend for technology development[J]. Synthetic Biology Journal,2021,2(5):764−777. doi: 10.12211/2096-8280.2021-057 ZHOU Zhengfu, PANG Yu, ZHANG Wei, et al. Recombinant expression of milk proteins and biosynthesis of animal-free milk: analysis on related patents and trend for technology development[J]. Synthetic Biology Journal, 2021, 2(5): 764-777. doi: 10.12211/2096-8280.2021-057
[15]	张艳敏. 对虾溶菌酶重组蛋白在大肠杆菌表达及包涵体复性研究[D]. 大连: 大连工业大学, 2014. ZHANG Yanmin. Recombinant expression of shrimp lysozyme in Escherichia coli and refolding of shrimp lysozyme inclusion bodies[D]. Dalian: Dalian Polytechnic University, 2014.
[16]	BASIONY M, OUYANG L, WANG D, et al. Optimization of microbial cell factories for astaxanthin production: Biosynthesis and regulations, engineering strategies and fermentation optimization strategies[J]. Synthetic and Systems Biotechnology,2022,7(2):689−704. doi: 10.1016/j.synbio.2022.01.002
[17]	朱明慧, 李晓静, 王浩民, 等. 原核和真核双表达载体的构建及功能分析[J]. 中国细胞生物学学报,2022,44(3):437−442. [ZHU Minghui, LI Xiaojing, WANG Haomin, et al. Construction and functional analysis of prokaryotic and eukaryotic dual expression vectors[J]. Chinese Journal of Cell Biology,2022,44(3):437−442. ZHU Minghui, LI Xiaojing, WANG Haomin, et al. Construction and functional analysis of prokaryotic and eukaryotic dual expression vectors[J]. Chinese Journal of Cell Biology, 2022, 44(3): 437–442.
[18]	LIU Y, DONG X, WANG B, et al. Food synthetic biology-driven protein supply transition: From animal-derived production to microbial fermentation[J]. Chinese Journal of Chemical Engineering,2021,30:29−36. doi: 10.1016/j.cjche.2020.11.014
[19]	陈爱亮. 一种酪蛋白多肽、多肽抗原、抗体、试纸条及其应. CN114716531A[P]. 2022-07-08. CHEN Ailiang. Casein polypeptide, polypeptide antigen, antibody, test strip and its application. CN114716531A[P]. 2022-07-08.
[20]	BAUMAN D E, MATHER I H, WALL R J, et al. Major advances associated with the biosynthesis of milk[J]. Journal of Dairy Science,2006,89(4):1235−1243. doi: 10.3168/jds.S0022-0302(06)72192-0
[21]	NIELSEN J, TILLEGREEN C B, PETRANOVIC D. Innovation trends in industrial biotechnology[J]. Trends Biotechnol,2022,40(10):1160−1172. doi: 10.1016/j.tibtech.2022.03.007
[22]	HETTINGA K, BIJL E. Can recombinant milk proteins replace those produced by animals?[J]. Curr Opin Biotechnol,2022,75:102690. doi: 10.1016/j.copbio.2022.102690
[23]	KEPPLER J K, HEYSE A, SCHEIDLER E, et al. Towards recombinantly produced milk proteins: Physicochemical and emulsifying properties of engineered whey protein beta-lactoglobulin variants[J]. Food Hydrocolloids,2021,110:106132.1−106132.14.
[24]	HANSSON L, BERGSTRÖM S, HERNELL O, et al. Expression of human milk beta-casein in Escherichia coli: Comparison of recombinant protein with native isoforms[J]. Protein Expression and Purification,1993,4(5):373−381. doi: 10.1006/prep.1993.1049
[25]	GEISTLINGER T, JHALA R, KRUEGER K P, et al. Food products comprising milk proteins and non-animal proteins, and methods of producing the same: US20190216106[P]. 2019-07-18.
[26]	庞雨. 牛乳蛋白在大肠杆菌中的异源表达和组合合成[D]. 北京: 中国农业科学院, 2021. PANG Yu. Heterologous expression and combinatorial synthesis of bovine milk proteins in Escherichia coli[J]. Beijing: Chinese Academy of Agricultural Sciences Thesis, 2021.
[27]	张齐, 崔金明, 蒙海林, 等. 7种牛奶蛋白基因在大肠杆菌中的异源表达[J]. 集成技术,2016,5(6):79−84. [ZHANG Qi, CUI Jinming, MENG Hailin, et al. Synthesis of seven milk proteins in Escherichia coli[J]. Journal of Integration Technology,2016,5(6):79−84. ZHANG Qi, CUI Jinming, MENG Hailin, et al. Synthesis of seven milk proteins in Escherichia coli[J]. Journal of Integration Technology, 2016, 5(6): 79-84.
[28]	邢芳芳, 印遇龙, 黄瑞林, 等. 重组牛乳铁蛋白素在大肠杆菌中的表达[J]. 食品科学,2008,29(04):221−224. [XING Fangfang, YIN Yulong, HUANG Ruilin, et al. Expression of recombinant lactoferricin B in E. coli[J]. Food Science,2008,29(04):221−224. XING Fangfang, YIN Yulong, HUANG Ruilin, et al. Expression of recombinant lactoferricin B in E. coli[J]. Food Science, 2008, 29(04): 221-224.
[29]	LE N T P, PHAN T T P, PHAN H T T, et al. Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis[J]. Biotechnology Reports,2022,35:e00754−760. doi: 10.1016/j.btre.2022.e00754
[30]	KHANI M H, BAGHERI M. Skimmed milk as an alternative for IPTG in induction of recombinant protein expression[J]. Protein expression and purification,2020,170:105593−105596. doi: 10.1016/j.pep.2020.105593
[31]	金亮. 牛乳铁蛋白在枯草芽孢杆菌中的高效表达及铁饱和度的理性改造[D]. 无锡: 江南大学, 2022. JIN Liang. High expression of bovine lactoferrin in Bacillus subtilis and rational design of its iron saturation[D]. Wuxi: Jiangnan University, 2022.
[32]	李秋凤, 陈静, 赵婧邑, 等. D-阿洛酮糖3-差向异构酶在大肠杆菌内的高效可溶性表达及发酵条件研究[J]. 食品工业科技,2022,43(22):136−143. [LI Qiufeng, CHEN Jing, ZHAO Jingyi, et al. Efficient soluble expression and fermentation conditions of D-allulose 3-epimerase in Escherichia coli[J]. Science and Technology of Food Industry,2022,43(22):136−143. LI Qiufeng, CHEN Jing, ZHAO Jingyi, et al. Efficient soluble expression and fermentation conditions of D-allulose 3-epimerase in Escherichia coli[J]. Science and Technology of Food Industry, 2022, 43(22): 136−143.
[33]	郭帅, 陈梦仟, 朱新术, 等. 大肠杆菌表达的重组琼胶酶包涵体的纯化与复性条件研究[J]. 基因组学与应用生物学,2018,37(3):1197−1202. [GUO Shuai, CHEN Mengqian, ZHU Xinshu, et al. Purification and renaturation of recombinant agarase from inclusion body expressed by Escherichia coli[J]. Genomics and Applied Biology,2018,37(3):1197−1202. GUO Shuai, CHEN Mengqian, ZHU Xinshu, et al. Purification and renaturation of recombinant agarase from inclusion body expressed by Escherichia coli[J]. Genomics and Applied Biology, 2018, 37(3): 1197-1202.
[34]	古淑青. 一种A1/A2β-酪蛋白的质谱检测方法: 中国, 108519484A[P]. 2018-09-11. GU Shuqing. A method for the detection of A1/A2 β-casein by mass spectrometry: China, 108519484A[P]. 2018-09-11.
[35]	孟鑫. 嗜盐四联球菌CICC10469butA生物信息学分析及其表达的研究[D]. 广州: 华南理工大学, 2016. MENG Xin. The bioinformatics analysis and the expression study of butA gene from Tetragenococcus halophilus CICC10469[D]. Guangzhou: South China University of Technology, 2016.
[36]	SU Y, ZOU Z, FENG S, et al. The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli[J]. Journal of Biotechnology,2007,129(3):373−382. doi: 10.1016/j.jbiotec.2007.01.015
[37]	王振, 邓超, 程咏梅, 等. cspA冷休克启动子-重组人bFGF表达载体的构建及在大肠杆菌中可溶性表达研究[J]. 工业微生物,2015,45(4):12−18. [WANG Zhen, DENG Chao, CHENG Yongmei, et al. Construction of cspA cold-shock promoter-recombinant human bFGF expression vector and its soluble expression in E. coli[J]. Industrial Microbiology,2015,45(4):12−18. WANG Zhen, DENG Chao, CHENG Yongmei, et al. Construction of cspA cold-shock promoter-recombinant human bFGF expression vector and its soluble expression in E. coli[J]. Industrial Microbiology, 2015, 45(4): 12-18.

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Preparation of β-Casein Based on Escherichia coli Expression System

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