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Microbiology and Biotechnology Letters


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Microbial Biotechnology (MB)  |  Protein Structure, Function, and Engineering

Microbiol. Biotechnol. Lett. 2021; 49(1): 75-87

Received: September 16, 2020; Accepted: December 24, 2020

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

Lita Meilina1,2, Sri Budiarti 2,3, Apon Zaenal Mustopa1*, Huda Shalahudin Darusman4, Lita Triratna1, Muhammad Ajietuta Nugraha5, Muhammad Sabiq Bilhaq6, and Ratih Asmana Ningrum1

1Research Center for Biotechnology, Indonesian Institute of Science (LIPI), Bogor, Indonesia 2School of Biotechnology, IPB University, Bogor, Indonesia 3Indonesia Research Center for Bioresources and Biotechnology, IPB University, Bogor, Indonesia 4Primate Study Center, IPB University, Bogor, Indonesia 5Departement of Biochemistry, Faculty of Mathematics and Natural Science, IPB University, Bogor, Indonesia 6Faculty of Technobiology, Sumbawa University of Technology, Sumbawa, Indonesia

Correspondence to :
Apon Zaenal Mustopa,

Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα- 2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp45 signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SPUsp45-IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC50 values of 33.22 μg/ml and 127.2 μg/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 μg/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 μg/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codonoptimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

Keywords: Colorectal cancer, pNZ8148, IFN&alpha,-2b, recombinant protein, Lactococcus lactis, WiDr

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