Cloning and expression of pigC gene in Escherichia coli

Do Minh Trung; Do Hai Quynh; Tran Viet Tien; Nguyen Duy Bac; Do Thi Tuyen; Nguyen Thuy Duong

doi:10.15625/1811-4989/16/4/13488

Author affiliations

Authors

Do Minh Trung
Do Hai Quynh
Tran Viet Tien
Nguyen Duy Bac
Do Thi Tuyen
Nguyen Thuy Duong

DOI:

https://doi.org/10.15625/1811-4989/16/4/13488

Keywords:

PigC, prodigiosin, Vietnam, MBC, MAP

Abstract

Prodigiosin (Pg), which is particularly of interest because of anticancer and antimicrobial activities, can be produced through the PigC-catalyzed condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Therefore, the PigC protein plays an important role in prodigiosin biosynthetic pathway. However, studies related to PigC protein have not been carried out in Vietnam yet. In this work, the pigC gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Using PCR and universal primers, we amplified a fragment of 3 kb covering entire coding region of the pigC gene from Serratia sp. strain M5. The pigC gene was inserted into pJET1.2 vector, and then transformed into E. coli DH10B. The sequence of a recombinant vector pJET1.2/pigC was evaluated by using whole colony PCR amplification. Sequence alignment results revealed that the obtained pigC gene possesses 71.5% and 75.4% of nucleotide identity in comparison with two strains, Serratia 39006 and Serratia sp. AS9 published in GenBank with their respective accession numbers of AJ833001 and CP002773. The recombinant vector pJET1.2/pigC was used to reamplify pigC, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing recombinant vector pET22b/pigC was expressed in the auto-induced medium. The presence of PigC protein in the lysate was identified as a 100 kDa band through Western Blot analysis using anti his-tag antibody. Afterward, the PigC protein was purified by Ni-NTA column, and its expression level was quantified through SDS-PAGE analysis. The results of our study provide a potential material for producing prodigiosin from recombinant protein in Vietnam.

Downloads

Download data is not yet available.

References

Chawrai SR, Williamson NR, Mahendiran T, Salmond GP, Leeper FJ (2012) Characterisation of PigC and HapC, the prodigiosin synthetases from Serratia sp. and Hahella chejuensis with potential for biocatalytic production of anticancer agents. Chem Sci 3(2): 447-454.

Chawrai SR, Williamson NR, Salmond GP, Leeper FJ (2008) Chemoenzymatic synthesis of prodigiosin analogues-exploring the substrate specificity of PigC. Chem Commun (16): 1862-1864.

Darshan N, Manonmani H (2015) Prodigiosin and its potential applications. J Food Sci Technol 52(9): 5393-5407.

Diaz-Ruiz C, Montaner B, Perez-Tomas R (2001) Prodigiosin induces cell death and morphological changes indicative of apoptosis in gastric cancer cell line HGT-1. Histol Histopathol 16(2): 415-421.

Domröse A, Klein AS, Hage-Hülsmann J, Thies S, Svensson V, Classen T, Pietruszka J, Jaeger K-E, Drepper T, Loeschcke A (2015) Efficient recombinant production of prodigiosin in Pseudomonas putida. Front Microbiol 6: 972.

Harris AK, Williamson NR, Slater H, Cox A, Abbasi S, Foulds I, Simonsen HT, Leeper FJ, Salmond GP (2004) The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species-and strain-dependent genome context variation. Microbiology 150(11): 3547-3560.

Klein AS, Domröse A, Bongen P, Brass HU, Classen T, Loeschcke A, Drepper T, Laraia L, Sievers S, Jaeger K-E (2017) New prodigiosin derivatives obtained by mutasynthesis in Pseudomonas putida. ACS Synth Biol 6(9): 1757-1765.

Liu P, Zhu H, Zheng G, Jiang W, Lu Y (2017) Metabolic engineering of Streptomyces coelicolor for enhanced prodigiosins (RED) production. Sci China Life Sci 60(9): 948-957.

Montaner B, Navarro S, Piqué M, Vilaseca M, Martinell M, Giralt E, Gil J, Pérez‐Tomás R (2000) Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines. Br J Pharmacol 131(3): 585-593.

Neupane S, Finlay RD, Alstrom S, Goodwin L, Kyrpides NC, Lucas S, Lapidus A, Bruce D, Pitluck S, Peters L, Ovchinnikova G, Chertkov O, Han J, Han C, Tapia R, Detter JC, Land M, Hauser L, Cheng JF, Ivanova N, Pagani I, Klenk HP, Woyke T, Hogberg N (2012a) Complete genome sequence of Serratia plymuthica strain AS12. Stand Genomic Sci 6(2): 165-173.

Neupane S, Finlay RD, Kyrpides NC, Goodwin L, Alström S, Lucas S, Land M, Han J, Lapidus A, Cheng J-F, Bruce D, Pitluck S, Peters L, Ovchinnikova G, Held B, Han C, Detter JC, Tapia R, Hauser L, Ivanova N, Pagani I, Woyke T, Klenk H-P, Högberg N (2012) Complete genome sequence of the plant-associated Serratia plymuthica strain AS13. Stand Genomic Sci 7(1): 22-30.

Neupane S, Högberg N, Alström S, Lucas S, Han J, Lapidus A, Cheng J, Bruce D, Goodwin L, Pitluck S (2012) Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9. Stand Genomic Sci 6: 54–62.

Nguyen HH, Nguyen HAK (2015) Bioefficacy of Serratia marcescens isolated from entomopathogenic nematodes (EPN) and their secondary metabolite prodigiosin against Spodoptera litura. Science and Technology Development 18 (2): 5-15.

Nguyen SLT, Le DQ (2015) Purification and antibacteria activity of anticancer agent prodigiosin from Serratia marcescens M10. Academia Journal of Biology 37: 210-216.

Rahul S, Chandrashekhar P, Hemant B, Bipinchandra S, Mouray E, Grellier P, Satish P (2015) In vitro antiparasitic activity of microbial pigments and their combination with phytosynthesized metal nanoparticles. Parasitol Int 64(5): 353-356.

Sambrook J, Russell DW, Russell DW (2001) Molecular Cloning. A Laboratory Manual, 3rd ed Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Suryawanshi RK, Patil CD, Koli SH, Hallsworth JE, Patil SV (2017) Antimicrobial activity of prodigiosin is attributable to plasma-membrane damage. Nat Prod Res 31(5): 572-577.

Tomás P, Ricardo E, Montaner B (2003) Effects of the proapoptotic drug prodigiosin on cell cycle-related proteins in Jurkat T cells. Histol Histopathol 18(2): 379-385.

Wang Z, Li B, Zhou L, Yu S, Su Z, Song J, Sun Q, Sha O, Wang X, Jiang W (2016) Prodigiosin inhibits Wnt/β-catenin signaling and exerts anticancer activity in breast cancer cells. Proc Natl Acad Sci USA 113(46): 13150-13155.

Williamson NR, Simonsen HT, Ahmed RA, Goldet G, Slater H, Woodley L, Leeper FJ, Salmond GP (2005) Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2‐methyl‐3‐n‐amyl‐pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces. Mol Microbiol 56(4): 971-989.

Yamamoto C, Takemoto H, Kuno K, Yamamoto D, Tsubura A, Kamata K, Hirata H, Yamamoto A, Kano H, Seki T (1999) Cycloprodigiosin hydrochloride, a new H+/Cl− symporter, induces apoptosis in human and rat hepatocellular cancer cell lines in vitro and inhibits the growth of hepatocellular carcinoma xenografts in nude mice. Hepatology 30(4): 894-902.

You Z, Liu X, Zhang S, Wang Y (2018a) Characterization of a prodigiosin synthetase PigC from Serratia marcescens jx-1 and its application in prodigiosin analogue synthesis. Biochem Eng J 134: 1-11.

You Z, Zhang S, Liu X, Wang Y (2018b) Enhancement of prodigiosin synthetase (PigC) production from recombinant Escherichia coli through optimization of induction strategy and media. Prep Biochem Biotechnol 48(3): 226-233.

Zhang J, Shen Y, Liu J, Wei D (2005) Antimetastatic effect of prodigiosin through inhibition of tumor invasion. Biochem Pharmacol 69(3): 407-414.