Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer Functionalities
Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expressio...
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MDPI AG
2024-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/3/1818 |
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| author | Chih-Ching Yen Pei-Ying Wu Huan Ou-Yang Hsiao-Ling Chen Kowit-Yu Chong Ro-Lin Chang Chuan-Mu Chen |
| author_facet | Chih-Ching Yen Pei-Ying Wu Huan Ou-Yang Hsiao-Ling Chen Kowit-Yu Chong Ro-Lin Chang Chuan-Mu Chen |
| author_sort | Chih-Ching Yen |
| collection | DOAJ |
| description | Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the <i>AOX1</i> promoter was replaced with a glucose-inducible <i>G1</i> promoter (P<sub>G1</sub>) to govern the expression of recombinant porcine LF (rpLF) in <i>Pichia pastoris</i> GS115. High-copy-number P<sub>G1</sub>-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe<sup>3+</sup>) and exhibited growth inhibition against various pathogenic microbes (<i>E. coli</i>, <i>S. aureus</i>, and <i>C. albicans</i>) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications. |
| first_indexed | 2024-03-08T03:54:46Z |
| format | Article |
| id | doaj.art-7ebac4da97164af094cfae27cd5399a1 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-08T03:54:46Z |
| publishDate | 2024-02-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-7ebac4da97164af094cfae27cd5399a12024-02-09T15:14:43ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-02-01253181810.3390/ijms25031818Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer FunctionalitiesChih-Ching Yen0Pei-Ying Wu1Huan Ou-Yang2Hsiao-Ling Chen3Kowit-Yu Chong4Ro-Lin Chang5Chuan-Mu Chen6Department of Internal Medicine, China Medical University Hospital, College of Health Care, China Medical University, Taichung 404, TaiwanDepartment of Life Sciences, Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, TaiwanDepartment of Life Sciences, Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, TaiwanDepartment of Biomedical Science, Da-Yeh University, Changhua 515, TaiwanDepartment of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan 333, TaiwanDepartment of Life Sciences, Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, TaiwanDepartment of Life Sciences, Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, TaiwanLactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the <i>AOX1</i> promoter was replaced with a glucose-inducible <i>G1</i> promoter (P<sub>G1</sub>) to govern the expression of recombinant porcine LF (rpLF) in <i>Pichia pastoris</i> GS115. High-copy-number P<sub>G1</sub>-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe<sup>3+</sup>) and exhibited growth inhibition against various pathogenic microbes (<i>E. coli</i>, <i>S. aureus</i>, and <i>C. albicans</i>) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.https://www.mdpi.com/1422-0067/25/3/1818lactoferrin<i>G1</i> promoter<i>Pichia pastoris</i>fermentationtangential-flow ultrafiltrationiron-binding |
| spellingShingle | Chih-Ching Yen Pei-Ying Wu Huan Ou-Yang Hsiao-Ling Chen Kowit-Yu Chong Ro-Lin Chang Chuan-Mu Chen Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer Functionalities International Journal of Molecular Sciences lactoferrin <i>G1</i> promoter <i>Pichia pastoris</i> fermentation tangential-flow ultrafiltration iron-binding |
| title | Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer Functionalities |
| title_full | Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer Functionalities |
| title_fullStr | Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer Functionalities |
| title_full_unstemmed | Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer Functionalities |
| title_short | Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in <i>Pichia pastoris</i>: Unveiling Antimicrobial and Anticancer Functionalities |
| title_sort | production of bioactive porcine lactoferrin through a novel glucose inducible expression system in i pichia pastoris i unveiling antimicrobial and anticancer functionalities |
| topic | lactoferrin <i>G1</i> promoter <i>Pichia pastoris</i> fermentation tangential-flow ultrafiltration iron-binding |
| url | https://www.mdpi.com/1422-0067/25/3/1818 |
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