Construction of pFUM003 expression vector with extracellular secretion properties

authors:

avatar A. li Javadmanesh , avatar bahareh pakbaten , avatar Reza Majidzadeh Heravi , * , avatar Hassan H Kermanshahi , avatar Mohammad Hadi Sekhavati

Koomesh: Vol.17, issue 4; 1017-1023
article type: issue_documents_importer
received: October 02, 2015
accepted: February 02, 2016

how to cite: Javadmanesh A L, pakbaten B, Majidzadeh Heravi R, H Kermanshahi H, Sekhavati M H. Construction of pFUM003 expression vector with extracellular secretion properties. koomesh. 2024;17(4):e151197. 

Abstract

Introduction: Probiotics are beneficial microorganisms that have been used in food production as well as health promotion over the centuries. Recombinant probiotics are used to express and transfer native or recombinant molecules to mucosal surface of digestive tract to promote efficiency in nutrition and health. Lactococcus lactis is a potential candidate for the production of useful biological proteins. Since there is not any vector nominated for this bacterium to show the extracellular protein secrition properties, therefore the aim of this study was to increase the efficiency of expression and secretion of recombinant proteins by adding transmission signal peptide usp45 to pBU003. Materials and Methods: usp45 signal peptide has 81 nucleotides and previous studies indicated that it can also cause increase in the gene expression. In this study, after synthesising usp45 signal sequence, it was ligated into pBU003. Then the vector was transformed to Ecoli (MC1061). The recombinant plasmid named pFUM003 after “plasmid Ferdowsi University of Mashhad 003”. Results: Sequencing of recombinant plasmid containing usp45 showed that the plasmid had correct construction and the inserted sequence had exact match with the reference sequence (EU382094.1) reported in NCBI;#39s GenBank. Conclusion: The recombinant plasmid was named pFUM003. This recombinant plasmid structurally was correct and can be used to transmit into probiotics make them to benefit from extracellular protein production properties.

References

  • 1.

    Craford JS. Probiotic in animal nutrition. Proceeding of Arcansas Nutrition Conference 1979; PP;45-55.

  • 2.

    Afshar Mazandaran N, Rajab A. Probiotics and their application in animal nutrition. Noorbakhsh publish 2002. (Persian).

  • 3.

    Deirde PL, Elke KA. The use and effects of lactic acid bacteria in malting and brewing with their relationships to antifungal activity myocotoxins and gushig: A Review. J Inst Brew 2004; 110: 163-180.

  • 4.

    Borerro J, jimenez J. Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria. J Biotenol 2011; 156: 76-86.

  • 5.

    Mierau I, Kleerebezem M. 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. J Appl Microbiol Biotechnol 2005; 68: 705-717.

  • 6.

    Le Loire Y, Azevedo V, Oliveira S, Freitas A, Miyoshi A, Bermdez-Humarn A, et al. Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production, 2005. Microbiol Cell Factories 2005, 4: 2.

  • 7.

    Majidzadeh R. Investigate the possibility of producing recombinant probiotic strains with the ability to degradation plant phytate in broiler chickens [dissertation]. Anim Sci Ferdowsi Univ 2010. (Persian).

  • 8.

    van der Vossen JM, van der Lelie D, Venema G. Isolation and characterization of streptococcus cremoris Wg2-Specific Promoters. Appl Environ Microbiol 1978; 53: 2452-2457.

  • 9.

    Lutful Kabir SM. The role of probiotics in the poultry industry. Int Mol science 2009; 10: 3531-3546.

  • 10.

    van Rooijen JR, Gasson MJ, de Vos WM. Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity. J Bacteriol 1992; 174: 2273-2280.

  • 11.

    Borerro J, Jimenez J. Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis. J Appl Genet Mol Biotechnol 2011; 89: 131-143.

  • 12.

    Leiloir Y, Nouaille S, Commissaire J, Brtigny L, Gruss A, Langella P. Signal peptide and propeptide optimization for heterologous protein secretion in lactococcus lactis. Appl Environ Microbiol 2001; 67: 4119-4127.

  • 13.

    Luerce TD, Azevedo MS, Leblance JG, Azevedo V, Miyoshi A, Pontes DS. Recombinant lactococcus lactis fails to secrete bovine chymosine. Bioengineered 2014; 5: 363-370.

  • 14.

    Shigemori S, Oshiro K, Wang P, Yamamoto Y, Wang Y, Sato T, et al. Generation of dipeptidyl peptidase-IV-inhibiting peptides from -Lactoglobulin secreted by lactococcus lactis. Biomed Res Intern 2014; 393598.

  • 15.

    Neef J, Milder JF, Koedijk D, Klaassense M, Heeziuse E, van Strijp J, et al. Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis. Appl Microbiol Biotechnol 2015; 99: 9037-9048.

  • 16.

    Higuchi R, Krummel B, Saiki R. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions". Nucleic Acids Res 1988; 16: 7351-7367.

  • 17.

    Sambrook J, Russell DW. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York, 2001.

  • 18.

    van Asseldonk M, Rutten G, Oteman M, Siezen RJ, de Vos WM, Simons G. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp, lactis. Gene 1990; 95: 155-160.