Open Journal Systems

Expression, Purification and Activity Study of Amuc_1100

Xiaohua Liang, Guangyu Long, Jiaojiao Sun, Qingqing Liu

Article ID: 1170
Vol 4, Issue 1, 2020, Article identifier:

VIEWS - 440 (Abstract) 225 (PDF)

Abstract

Objective: To construct prokaryotic expression vector of Amuc_1100 gene, the ELP (ELSTIN-LIKE PROTEIN) is used as purification label to express fusion with the target protein and detect the biological activity of recombinant protein at cell level and animal level. Methods: the recombinant plasmid pET28a-Amuc_1100-ELP is designed and synthesized, and transformed into e. coli competent cell bl21 (de3) to induce expression. The recombinant egg was isolated and purified by the reversible phase change cycling (ITC) characteristic of ELP10 white. At the cellular level, PBMCs cells from mice were extracted to detect the expression level of IL-10. At the animal level, obese mice induced by gastric gavage with high fat detected the improvement effect of the protein on mouse metabolism. Results: The recombinant protein with the expected molecular weight of 60 kDa was expressed by E.coli expression system. After circulating through ITC, the recombinant protein with high purity was obtained. The protein increased the expression level of IL-10 in PBMCs cells, and significantly improved the obesity and metabolic disorders induced by high-fat diet in mice. Conclusion: The recombinant protein expression plasmid pET28a-Amuc_1100-ELP was successfully constructed. After expression and purification, the recombinant protein expression plasmid has high purity and has 15 biologically active recombinant protein. This study laid a foundation for the industrial production and clinical application of Amuc_1100.


Keywords

Molecular Biology; Escherichia Coli; Recombinant Protein; ITC cycle; Metabolic Syndrome

Full Text:

PDF


Included Database


References

Turek FW, Joshu C, Kohsaka A, et al. Obesity and metabolic syndrome in circadian clock mutant mice[J]. Science, 2005, 308(5724): 1043-1045.

Eckel RH, Alberti K, Grundy SM, et al. The metabolic syndrome[J]. The Lancet, 2010, 375(9710): 181-183.

Backhed F, Ding H, Wang T et al.The gut microbiota as an environmental factor that regulates fat storage[J]. Proc Natl Acad Sci USA,2004,101:15718-15723.

Le Chatelier E, Nielsen T, Qin JJ, Prifti E, Hildebrand F, Falony G, et al. Richness of human gut microbiome correlates with metabolic markers[J]. Nature, 2013, 500(7464): 541-7.

Karlsson FH, Tremaroli V, Nookaew I, Bergstrom G, Behre CJ, Fagerberg B, et al. Gut metagenome in European women with normal, impaired and diabetic glucose control[J]. Nature, 2013, 498(7452): 99-23.

Cho I, Blaser MJ. The human microbiome: at the interface of health and disease[J]. Nature Reviews Genetics, 2012, 13(4): 260-270.

Tremaroli V, Bäckhed F. Functional interactions between the gut microbiota and host metabolism[J]. Nature, 2012, 489(7415): 242-249.

Vrieze A, van Nood E, Holleman F, Salojärvi J, Kootte RS, Bartelsman JFWM, et al. Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome[J]. Gastroenterology, 2012,143(4): 913-17.

Nieuwdorp M, Gilijamse PW, Pai N, et al. Role of the microbiome in energy regulation and metabolism[J]. Gastroenterology, 2014, 146(6): 1525-1533.

Li J, Lin SQ, Vanhoutte PM, Woo CW, Xu AM. Akkermansia Muciniphila Protects Against Atherosclerosis by Preventing Metabolic Endotoxemia-Induced Inflammation in Apoe(-/-) Mice[J]. Circulation, 2016, 133(24): 2434-63.

Schneeberger M, Everard A, Gomez-Valades AG, Matamoros S, Ramirez S, Delzenne NM et al. Akkermansia muciniphila inversely correlates with the onset of inflammation, altered adipose tissue metabolism and metabolic disorders during obesity in mice[J]. Sci Rep. 2015, 5: 166-43.

Zhao S, Liu W, Wang J, Shi J, Sun Y, Wang W, Ning G, Liu R, Hong J. Akkermansia muciniphila improves metabolic profiles by reducing inflammation in chow diet-fed mice[J]. J. Mol. Endocrinol, 2017, 58, 1−14.

Plovier H, Everard A, Druart C, Depommier C, Van Hul M, Geurts L, et al. A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice[J]. Nature Medicine, 2017, 23(1): 107-13.

Anhe FF, Marette A. A microbial protein that alleviates metabolic syndrome[J]. Nature Medicine, 2017, 23(1): 11-2.

Ottman N, Reunanen J, Meijerink M, Pietila TE, Kainulainen V, Klievink J, et al. Pili-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function[J]. Plos One, 2017, 12(3).

Qi Hao, Liu Xinli. Research progress of Escherichia coli expression system and yeast expression system[J]. Anhui Agricultural Sciences, 2016, 44(17): 46-52.

Gao Yuhui, Deng Weiwei. Prokaryotic expression and one-step preparation of soluble diphtheria toxin mutant CRM197[J]. Basic Medicine and Clinic, 2018.

(06): 815-20.

Fletcher EE, Yan DD, Kosiba AA, Zhou Y, Shi HF. Biotechnological applications of elastin-like polypeptides and the inverse transition cycle in the pharmaceutical industry[J]. Protein Expres Purif, 2019, 153: 114-20.

Hu F, Ke T, Li X, Mao PH, Jin X, Hui FL, et al. Expression and Purification of an Antimicrobial Peptide by Fusion with Elastin-like Polypeptides in Escherichia coli[J]. Appl Biochem Biotech, 2010, 160(8): 2377-87.

Lim DW, Trabbic-Carlson K, Mackay JA, et al. Improved non-chromatographic purification of a recombina protein by cationic elastin-like polypeptidesnt[J]. Biomacromolecules, 2007, 8(5): 1417-1427.

Matsumoto R, Nara R, Andou T, Mie M, Kobatake E. Targeting of EGF-displayed protein nanoparticles with anticancer drugs[J]. J Biomed Mater Res B. 2014; 102(8): 1792-8.

Alhawi M, Stewart J, Erridge C, Patrick S, Poxton IR. Bacteroides fragilis signals through Toll-like receptor (TLR) 2 and not through TLR4. Journal of medical microbiology. 2009; 58(Pt 8): 1015-22.

Sezer H. Insulin resistance, obesity and lipotoxicity. Obesity and Lipotoxicity: Springer; 2017. p. 277–304


DOI: http://dx.doi.org/10.18063/gse.v4i1.1170
(440 Abstract Views, 225 PDF Downloads)

Refbacks

  • There are currently no refbacks.


Copyright (c) 2020 Xiaohua Liang, Guangyu Long, Jiaojiao Sun, Qingqing Liu

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.