重组牛乳铁蛋白功能片段在毕赤酵母中的表达及高密度发酵
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摘要
该研究合成了密码子优化后的牛乳铁蛋白功能片段(bovine lactoferrin functional fragment,Blf Ff),转入毕赤酵母GS115中重组表达并筛选抗性菌株,通过镍亲和层析纯化Blf Ff,以Western Blot、液质联用和ELISA对重组蛋白进行鉴定及检测。比较了5 L发酵罐生产中3种不同高密度发酵培养基对Blf Ff生产的影响。结果表明:重组转化子正确表达了Blf Ff。镍亲和层析中,150 mmol/L咪唑洗脱可得到电泳纯37 k Da的目标条带。在3种高密度发酵培养基中,RDM最优,诱导48 h,菌体湿细胞密度达到297 g/L,Blf Ff表达量为38. 1 mg/L。
This study aimed to express bovine lactoferrin functional fragments( BlfFf) in Pichia pastoris and investigate high-cell-density fermentation. The codon-optimized BlfFf was synthesized and transferred into P. pastoris GS115 for recombinant expression,followed by resistant strain screening. The BlfFf was purified with Ni affinity chromatography,and identified and determined by Western Blot,LC-MS,and ELISA assays. The effects of three different high-cell-density fermentation media on BlfFf production in a 5 L fermentor were compared. The results showed that the engineered yeast expressed BlfFf correctly,and a 37 kDa electrophoretic pure target band was obtained by elution with 150 mmol/L imidazole. Moreover,RDM was found to be the optimal culture medium. After 48 h induction,the wet cell density reached 297 g/L and the yield reached 38. 1 mg/L. In conclusion,the recombinant P. pastoris was successfully constructed to produce BlfFf in RDM medium for high-cell-density fermentation,which shows its industrial application potentials.
This study aimed to express bovine lactoferrin functional fragments( BlfFf) in Pichia pastoris and investigate high-cell-density fermentation. The codon-optimized BlfFf was synthesized and transferred into P. pastoris GS115 for recombinant expression,followed by resistant strain screening. The BlfFf was purified with Ni affinity chromatography,and identified and determined by Western Blot,LC-MS,and ELISA assays. The effects of three different high-cell-density fermentation media on BlfFf production in a 5 L fermentor were compared. The results showed that the engineered yeast expressed BlfFf correctly,and a 37 kDa electrophoretic pure target band was obtained by elution with 150 mmol/L imidazole. Moreover,RDM was found to be the optimal culture medium. After 48 h induction,the wet cell density reached 297 g/L and the yield reached 38. 1 mg/L. In conclusion,the recombinant P. pastoris was successfully constructed to produce BlfFf in RDM medium for high-cell-density fermentation,which shows its industrial application potentials.
引文
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[5] WANG X,WANG X,HAO Y,et al. Research and development on lactoferrin and its derivatives in China from2011-2015[J]. Biochemistry and Cell Biology,2017,95(1):162-170.
[6] JENSSEN H,SANDVIK K,ANDERSEN J H,et al. Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin[J]. Antiviral Research,2008,79(3):192-198.
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[8] BLANCA I F,NORBERTO V G,TANIA S C,et al.High-level expression of recombinant bovine lactoferrin in Pichia pastoris with antimicrobial activity[J]. International Journal of Molecular Sciences,2016,17(6):902.
[9]郭爱珍,吕自力,钟浩,等.牛乳铁蛋白肽及其衍生肽生物活性与研究进展[J].中国乳品工业,2016,44(5):22-27.
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[11] LEVAY P F,VILJOEN M. Lactoferrin:a general review[J]. Haematologica,1995,80(3):252-267.
[12] TANG Xiangshan,TANG Zhiru,WANG Shengping,et al. Expression,purification,and antibacterial activity of bovine lactoferrampin–lactoferricin in Pichia pastoris[J]. Protein Expression and Purification,2010,73(2):132-139.
[13] GONZLEZ-CHVEZ S A,ARVALO-GALLEGOS S,RASCN-CRUZ Q. Lactoferrin:structure,function and applications[J]. International Journal of Antimicrobial Agents,2009,33(4):301. e1-301. e8.
[14] TANG Xiangshan,SHAO Hua,TANG Zhiru,et al. Dietary supplementation with bovine lactoferrampin-lactoferricin produced by Pichia pastoris fed-batch fermentation affects intestinal microflora in weaned piglets[J]. Applied Biochemistry&Biotechnology,2012,168(4):887-898.
[15] BOLSCHER J G M,ADAO R,NAZMI K,et al. Bactericidal activity of lfchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides[J]. Biochimie, 2009, 91(1):123-132.
[16]郭东华,孙东波,孙斌,等.牛乳铁蛋白肽基因(Lfcin B)的合成及其在大肠杆菌中的表达[J].农业生物技术学报,2009,17(3):451-454.
[17] LIANG Shuli,WANG Bin,PAN Li,et al. Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing[J]. BMC Genomics,2012,13(1):738.
[18]马银鹏,王玉文,党阿丽,等.毕赤酵母表达系统研究进展[J].黑龙江科学,2013,4(9):27-31.
[19]江鹏,汤斌.蚓激酶基因在毕赤酵母中的表达及其发酵条件优化[J].食品与发酵工业,2018,44(10):79-83.
[20]王鑫,金鹏,宋鹏,等.黑曲霉酸性蛋白酶EXPA的克隆表达与酶学性质解析[J].食品与发酵工业,2019,45(3):40-46.
[21] BARRIG N J M,VALERO F,MONTESINOS J L. A macrokinetic model-based comparative meta-analysis of recombinant protein production by Pichia pastoris under AOX1 promoter[J]. Biotechnology and Bioengineering,2015,112(6):1 132-1 145.
[22] CHAHARDOOLI M,NIAZI A,ARAM F,et al. Expression of recombinant arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification[J]. Journal of the Science of Food and Agriculture,2016,96(2):569-575.
[23] YEN C C. Expression of lactoferrin in Pichia pastoris induction by glucose in a modified g1 promoter system and its antimicrobial and antitumor activities[J]. Respirology,2017,22:129-130.
[24] LI J,ZHU W,LUO M,et al. Molecular cloning,expression and purification of lactoferrin from tibetan sheep mammary gland using a yeast expression system[J]. Protein Expression and Purification,2015,109:35-39.
[25] D'ANJOU M C,DAUGULIS A J. Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast[J]. Biotechnology Letters,2000,22(5):341-346.
[26] BARTLETT M C,KUO A,ROUTENBERG L K,et al.Development of a general defined medium for Pichia pastoris[J]. Biotechnology and Bioengineering,2018,115(1):103-113.
[2]朱艳萍,滕达,田子罡.乳铁蛋白分子结构及其抗菌机制[J].生物技术通报,2010(6):37-42.
[3]杨鹏华,倪凤娥.常乳中牛乳铁蛋白的纯化及抗菌活性研究[J].安徽农业科学,2009,37(16):7 351-7 352.
[4] BELLAMY W,TAKASE M,WAKABAYASHI H,et al.Antibacterial spectrum of lactoferricin B,a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin[J]. Journal of Applied Microbiology,1992,73(6):472-479.
[5] WANG X,WANG X,HAO Y,et al. Research and development on lactoferrin and its derivatives in China from2011-2015[J]. Biochemistry and Cell Biology,2017,95(1):162-170.
[6] JENSSEN H,SANDVIK K,ANDERSEN J H,et al. Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin[J]. Antiviral Research,2008,79(3):192-198.
[7] GROVES M L. The isolation of a red protein from milk[J]. Journal of the American Chemical Society,1960,82(13):3 345-3 350.
[8] BLANCA I F,NORBERTO V G,TANIA S C,et al.High-level expression of recombinant bovine lactoferrin in Pichia pastoris with antimicrobial activity[J]. International Journal of Molecular Sciences,2016,17(6):902.
[9]郭爱珍,吕自力,钟浩,等.牛乳铁蛋白肽及其衍生肽生物活性与研究进展[J].中国乳品工业,2016,44(5):22-27.
[10] JOHANSONB. Isolation of an iron-containing red protein from milk[J]. Acta Chem Scand,1960,14(2):510-512.
[11] LEVAY P F,VILJOEN M. Lactoferrin:a general review[J]. Haematologica,1995,80(3):252-267.
[12] TANG Xiangshan,TANG Zhiru,WANG Shengping,et al. Expression,purification,and antibacterial activity of bovine lactoferrampin–lactoferricin in Pichia pastoris[J]. Protein Expression and Purification,2010,73(2):132-139.
[13] GONZLEZ-CHVEZ S A,ARVALO-GALLEGOS S,RASCN-CRUZ Q. Lactoferrin:structure,function and applications[J]. International Journal of Antimicrobial Agents,2009,33(4):301. e1-301. e8.
[14] TANG Xiangshan,SHAO Hua,TANG Zhiru,et al. Dietary supplementation with bovine lactoferrampin-lactoferricin produced by Pichia pastoris fed-batch fermentation affects intestinal microflora in weaned piglets[J]. Applied Biochemistry&Biotechnology,2012,168(4):887-898.
[15] BOLSCHER J G M,ADAO R,NAZMI K,et al. Bactericidal activity of lfchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides[J]. Biochimie, 2009, 91(1):123-132.
[16]郭东华,孙东波,孙斌,等.牛乳铁蛋白肽基因(Lfcin B)的合成及其在大肠杆菌中的表达[J].农业生物技术学报,2009,17(3):451-454.
[17] LIANG Shuli,WANG Bin,PAN Li,et al. Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing[J]. BMC Genomics,2012,13(1):738.
[18]马银鹏,王玉文,党阿丽,等.毕赤酵母表达系统研究进展[J].黑龙江科学,2013,4(9):27-31.
[19]江鹏,汤斌.蚓激酶基因在毕赤酵母中的表达及其发酵条件优化[J].食品与发酵工业,2018,44(10):79-83.
[20]王鑫,金鹏,宋鹏,等.黑曲霉酸性蛋白酶EXPA的克隆表达与酶学性质解析[J].食品与发酵工业,2019,45(3):40-46.
[21] BARRIG N J M,VALERO F,MONTESINOS J L. A macrokinetic model-based comparative meta-analysis of recombinant protein production by Pichia pastoris under AOX1 promoter[J]. Biotechnology and Bioengineering,2015,112(6):1 132-1 145.
[22] CHAHARDOOLI M,NIAZI A,ARAM F,et al. Expression of recombinant arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification[J]. Journal of the Science of Food and Agriculture,2016,96(2):569-575.
[23] YEN C C. Expression of lactoferrin in Pichia pastoris induction by glucose in a modified g1 promoter system and its antimicrobial and antitumor activities[J]. Respirology,2017,22:129-130.
[24] LI J,ZHU W,LUO M,et al. Molecular cloning,expression and purification of lactoferrin from tibetan sheep mammary gland using a yeast expression system[J]. Protein Expression and Purification,2015,109:35-39.
[25] D'ANJOU M C,DAUGULIS A J. Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast[J]. Biotechnology Letters,2000,22(5):341-346.
[26] BARTLETT M C,KUO A,ROUTENBERG L K,et al.Development of a general defined medium for Pichia pastoris[J]. Biotechnology and Bioengineering,2018,115(1):103-113.