Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)

Olfa Mega, Cece Sumantri, Irma Isnafia Arief, Cahyo Budiman


Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico.  pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively.  Molecular weight was validated using SDS-PAGE and software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C.  As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.


E. coli; gene expression; L. plantarum; Lon-like protease.

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Arief, I.I., Budiman, C., Jenie, B., Andreas. E., Yuneni, A. 2015. Plantarisin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity against Staphylococcus aureus. Beneficial Microbes. 6:603-613.

Barnum, S.R. 2005. Biotechnology an Introduction. International Student Edition. Ed ke-2. Belmont, Thmpson Brooks/Cole.

Brown, T.A. 2001. Gene Cloning and DNA Analysis: An Introduction. 4th ed, Blackwell Science Ltd., USA.

Carbone, A., Zinovyev, A., Képès, F. 2003. Codon adaptation index as a measure of dominating codon bias. Bioinformatics. 19:2005-2015.

Gaffar, B. 2010. Produksi Protein Rekombinan dalam Sistem Ekspresi Pichia pastoris. Unpad Press, Bandung.

GenScript. 2019. Optimum GeneTM Codon : Optimization Analysis. GenScript USA Inc, Centennial Ave, USA.

Hughes, R.A., Miklos, A.E., Ellington, A.D. 2011. Gene synthesis: methods and applications. Methods Enzymol. 498:277-309. doi: 10.1016/B978-0-12-385120-8.00012-7.

Kane, J.F. 1995. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coil. J. Current Opin Biotech. 6:494-500.

Kang, Y.S., Song, J.A., Han, K.Y., Lee, J. 2015. Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins. J. Biotech. 194: 39-47.

Kapetanovic, I.M. 2008. Computer-aided drug discovery and development (CADDD): in silico-chemico-biological approach. Chem Biol Interact. 171(2): 165–176. doi:10.1016/j.cbi.2006.12.006.

Laemmi, U.K. 1970. Cleave of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-685.

Li, Q., Yi, L., Marek, P., Iverson, B.L. 2013. Commercial proteases: Present and future. FEBS Letters.doi: 10.1016/j.febslet.2012.12.019.

Melderen, L.V., Aertsen, A. 2009.Regulation and quality control by Lon-dependent proteolysis. Research in Microbiology. 160:645-651

Nakamura, N., Ikemura ,T. 1995. FOP (Frequency of Optimal Codons Usage). WWW Service with Its Distribution Analysis. National Institute of Genetics and Graduate University of Advanced Studies. Zhizuoka, Jepang.

Newman, M., Strzelecka, T., Dorner, L.F., Schildkraut, I., Aggarwal, A.K. 1994. Structure of endonuclease BamH1 and its relationship to EcoR1. Nature. 368: 660-664.

Novelli, P.K., Barros, M.M., Fleuri, L.F. 2016. Novel inexpensive fungi protease: production by solid state fermentation and characterization. Food Chem. 198:119-124.doi:10.1016/j.foodchem2015.11.089.

Pinti, M., Gibellini, L., Nasi, M., De Biasi, S., Bortolotti, C.A., Iannone, A., Cossarizza, A. 2016. Emerging role of Lon protease as a master regulator of mitochondrial functions. Biochimica et Biophysica Acta 1857 : 1300–1306.

Rosen, R., Biran, D., Gur, E., Becher, D., Hecker, M., Ron, E.Z. 2002. Protein aggregation in Escherichia coli: Role of proteases. FEMS Microbiol. Lett. 207:9–12.

Sambrook, J., Russel, D.W. 2001. Expression of cloned genes in Escherichia coli (chapter 15). In: Molecular Cloning: A Laboratory Manual. 3th Ed. Cold Spring Harbor Laboratory Press. New York.

Stanbury, P.F., Whitaker, A., Hall, S.J. 2016. Principles of Fermentation Technology. 3¬th Ed. Heinemann, Butterworth.

Thies, F. L., Hartung, H.P., Giegerich, G.1998. Cloning and expression of the Campylobacter jejuni lon gene detected by RNA arbitrarily primed PCR. FEMS Microbiology Letters, 165 :329-334.

Uttatree, S., Charoempanic, J. 2016. Isolation and characterization of a broad pH- and temperature –active, solven and sulfactant stable protease from a new strain of Bacillus subtilis. Biocatalysis and Agricultural Biotechnology. 8:32-38. doi:10.1016/j.bcab.2016.08.003.

Yaraguppi, D.A., Udapudi, B.B., Patil, L.R., Hombalimath, V.S., Shet, A.R. 2012. In-silico analysis for predicting protein ligand interaction for snake venom protein. J. Adv. Bioinform. Appl Research.3(3): 345-356.

Yoon, S.I.I., Walter, M.R. 2007. Identification and characterization of a +1 frameshift observed during the expression of Epstein-Barr virus IL-10 in Escherichia coli. Protein Expression and Purification 53:132–137.

Yuwono, T. 2008. Bioteknologi Pertanian. Gadjah Mada University Press, Yogyakarta.

Watson, R.J., Schildkraut, I., Qiang, B.Q., Martin, S.M., Visentin, L.P. 1982. NdeI: a restriction endonuclease from Neisseria denitrificans which cleaves DNA at 5‘-CATATG-3’ sequences. Febs Letters. 150(1):114-116.



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