Optimized Yeast Expression of Canine Interferon-α Using a Combined Strategy

doi:10.13523/j.cb.2107010

China Biotechnology

2022, Vol. 42

Issue (1/2): 88-95 DOI: 10.13523/j.cb.2107010

Orginal Article

Optimized Yeast Expression of Canine Interferon-α Using a Combined Strategy

LI Ding^1,^2,³,LI Lan^1,^2,³,AN Yun-fei⁴,BI Zhen-wei⁵,YU Xiao-ming^1,^2,³,CHEN Jin^1,^2,³,ZHENG Qi-sheng^1,^2,^3,^**(

)

1 Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
2 Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose, Yangzhou University, Yangzhou 225009, China
3 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology, Nanjing 210014, China
4 College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
5 Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

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Abstract

Canine interferon-α(CaIFN) was expressed in Pichia pastoris in the previous studies, which had high biological activity and relatively low yield. Therefore, increasing the yield of recombinant protein is the key task to promote the application of CaIFN. Yeast transformants containing 1, 2, 4, 6, and 8 copies of CaIFN gene were generated, and the yield of KM-6CaIFN was the highest, which was 200.6% higher than that of KM-1CaIFN. 8 molecular chaperones were co-expressed with CaIFN, and the Hac protein could increase the target protein yield of KM-6CaIFN and KM-8CaIFN by 32.1% and 113.1%, respectively. More copies of CaIFN were integrated into KM-8CaIFN-Hac strain. SDS-PAGE analysis showed that the yield of KM-12CaIFN-Hac was 1.33 times that of KM-8CaIFN-Hac, and 5.61 times that of KM-1CaIFN, respectively. By comparing protein bands with serially dilution BSA, the yield of KM-12CaIFN-Hac was estimated to be about 581 mg/L, which is the highest value in P. pastoris reported so far.

Key words： CaIFN Pichia pastoris Recombinant protein yield Multi-copy Molecular chaperones

Received: 03 July 2021 Published: 03 March 2022

ZTFLH:

Q819

Corresponding Authors: Qi-sheng ZHENG E-mail: njcvc1302@163.com

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Cite this article:

LI Ding,LI Lan,AN Yun-fei,BI Zhen-wei,YU Xiao-ming,CHEN Jin,ZHENG Qi-sheng. Optimized Yeast Expression of Canine Interferon-α Using a Combined Strategy. China Biotechnology, 2022, 42(1/2): 88-95.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2107010 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I1/2/88

Table 1 The primers used in this study

Table 2 The molecular chaperones used in this study

Fig.1 SDS-PAGE analysis (a) and Western blot analysis (b) of supernatant of KM-1CaIFN Lane M: Protein molecular weight marker; Lane 1: Supernatant of KM-1CaIFN; Lane 2: Endo Hf-treated supernatant of KM-1CaIFN. 10 μL sample was loaded for each lane

Fig.2 Verification of recombinant expression vectors Lane M: DNA molecular weight marker; Lane 1: pMCO-AOXα-CaIFN; Lane 2: pMCO-AOXα-2CaIFN; Lane 3: pMCO-AOXα-4CaIFN; Lane 4: pMCO-AOXα-6CaIFN; Lane 5: pMCO-AOXα-8CaIFN

Table 3 The copy number analysis of the yeast transformants

Fig.3 Effect of gene copy number on CaIFN production (a) SDS-PAGE analysis of supernatants of yeast transformants (b)Semi-quantitative analysis Lane M: Protein molecular weight marker; Lane 1: KM-1CaIFN; Lane 2: KM-2CaIFN; Lane 3: KM-4CaIFN; Lane 4: KM-6CaIFN; Lane 5: KM-8CaIFN. 10 μL sample was loaded for each lane

Fig.4 Effect of co-expressing molecular chaperones on CaIFN production in KM-6CaIFN strain (a) SDS-PAGE analysis of supernatants of yeast transformants (b)Semi-quantitative analysis Lane M: Protein molecular weight marker; Lane 1: KM-6CaIFN; Lane 2: KM-6CaIFN-Hac; Lane 3: KM-6CaIFN-Fhl; Lane 4: KM-6CaIFN-INO; Lane 5: KM-6CaIFN-YDJ; Lane 6: KM-6CaIFN-SSA4; Lane 7: KM-6CaIFN-BiP; Lane 8: KM-6CaIFN-PDI; Lane 9: KM-6CaIFN-Erv. 10 μL sample was loaded for each lane

Fig.5 Effect of co-expressing molecular chaperones on CaIFN production in KM-8CaIFN strain (a) SDS-PAGE analysis of supernatants of yeast transformants (b)Semi-quantitative analysis Lane M: Protein molecular weight marker; Lane 1: KM-8CaIFN; Lane 2: KM-8CaIFN-Hac; Lane 3: KM-8CaIFN-Fhl; Lane 4: KM-8CaIFN-INO; Lane 5: KM-8CaIFN-YDJ; Lane 6: KM-8CaIFN-SSA4; Lane 7: KM-8CaIFN-BiP; Lane 8: KM-8CaIFN-PDI; Lane 9: KM-8CaIFN-Erv. 10 μL sample was loaded for each lane

Fig.6 Effect of increasing gene copy number on CaIFN production in the strains co-expressing Hac protein (a) SDS-PAGE analysis of supernatants of yeast transformants (b)Semi-quantitative analysis Lane M: Protein molecular weight marker; Lane 1: KM-1CaIFN; Lane 2: KM-6CaIFN; Lane 3: KM-6CaIFN-Hac; Lane 4: KM-8CaIFN; Lane 5: KM-8CaIFN-Hac; Lane 6: KM-12CaIFN-Hac; Lane 7: KM-14CaIFN-Hac; Lane 8: KM-16CaIFN-Hac. 10 μL sample was loaded for each lane

Fig.7 Quantification of CaIFN protein in the supernatant of KM-1CaIFN and KM-12CaIFN-Hac strain Lane M: Protein molecular weight marker; Lane 1-7: Serially diluted BSA at 25, 50, 100, 200, 400, 600, 800 mg/L; Lane 8: KM-1CaIFN; Lane 9: KM-12CaIFN-Hac. 20 μL sample was loaded for each lane in the SDS-PAGE assay


[1]	昝芳, 白海霞, 崔君. 天津地区犬常见传染病的调查及防控措施. 天津农学院学报, 2017, 24(4):90-93.

[1]	Zan F, Bai H X, Cui J. Investigation and prevention control measures of common infectious diseases in canine in Tianjin area. Journal of Tianjin Agricultural University, 2017, 24(4):90-93.

[2]	Borden E C. Interferons: rationale for clinical trials in neoplastic disease. Annals of Internal Medicine, 1979, 91(3):472. pmid: 382941

[3]	Tompkins W A. Immunomodulation and therapeutic effects of the oral use of interferon-alpha: mechanism of action. Journal of Interferon & Cytokine Research, 1999, 19(8):817-828.

[4]	Carvalho O V, Saraiva G L, Ferreira C G T, et al. In-vitro antiviral efficacy of ribavirin and interferon-alpha against canine distemper virus. Canadian Journal of Veterinary Research, 2014, 78(4):283-289.

[5]	Werten M W, van den Bosch T J, Wind R D, et al. High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast (Chichester, England), 1999, 15(11):1087-1096. doi: 10.1002/(ISSN)1097-0061

[6]	Li D, Zhang B, Li S T, et al. A novel vector for construction of markerless multicopy overexpression transformants in Pichia pastoris. Frontiers in Microbiology, 2017, 8:1698. doi: 10.3389/fmicb.2017.01698

[7]	曹瑞兵, 王艳, 魏建超, 等. 犬α1干扰素在毕赤酵母中的分泌表达及其抗病毒活性研究. 南京农业大学学报, 2008, 31(4):107-112.

[7]	Cao R B, Wang Y, Wei J C, et al. Secreted expression of canine interferon alpha subtype 1 in Pichia pastoris and its antiviral activity. Journal of Nanjing Agricultural University, 2008, 31(4):107-112.

[8]	Li D, Zhang H M, Yang L, et al. Surface display of classical swine fever virus E2 glycoprotein on gram-positive enhancer matrix (GEM) particles via the SpyTag/SpyCatcher system. Protein Expression and Purification, 2020, 167:105526. doi: 10.1016/j.pep.2019.105526

[9]	Li D, Wu J C, Chen J, et al. Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris. Protein Expression and Purification, 2020, 167:105527. doi: 10.1016/j.pep.2019.105527

[10]	Guerfal M, Ryckaert S, Jacobs P P, et al. The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microbial Cell Factories, 2010, 9:49. doi: 10.1186/1475-2859-9-49 pmid: 20591165

[11]	Zhao H, Blazanovic K, Choi Y, et al. Gene and protein sequence optimization for high-level production of fully active and aglycosylated lysostaphin in Pichia pastoris. Applied and Environmental Microbiology, 2014, 80(9):2746-2753. doi: 10.1128/AEM.03914-13

[12]	Zhu T, Guo M, Tang Z, et al. Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris. Journal of Applied Microbiology, 2009, 107(3):954-963. doi: 10.1111/j.1365-2672.2009.04279.x pmid: 19486418

[13]	Delic M, Valli M, Graf A B, et al. The secretory pathway: exploring yeast diversity. FEMS Microbiology Reviews, 2013, 37(6):872-914. doi: 10.1111/1574-6976.12020

[14]	Zheng X Y, Zhang Y M, Zhang X Y, et al. Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins. Microbial Cell Factories, 2019, 18(1):207. doi: 10.1186/s12934-019-1256-0

[15]	Chang H J, Jones E W, Henry S A. Role of the unfolded protein response pathway in regulation of INO1 and in the sec14 bypass mechanism in Saccharomyces cerevisiae. Genetics, 2002, 162(1):29-43. doi: 10.1093/genetics/162.1.29 pmid: 12242221

[16]	Yu P, Zhu Q, Chen K F, et al. Improving the secretory production of the heterologous protein in Pichia pastoris by focusing on protein folding. Applied Biochemistry and Biotechnology, 2015, 175(1):535-548. doi: 10.1007/s12010-014-1292-5

[17]	Yin Y, Garcia M R, Novak A J, et al. Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic Reticulum. PLoS Biology, 2018, 16(8):e2005140. doi: 10.1371/journal.pbio.2005140

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