Method for the mass expression of an antimicrobial peptide...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...

Reexamination Certificate

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C435S071100, C435S071200, C435S071300

Reexamination Certificate

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08003348

ABSTRACT:
The present invention relates to a gene construct which is capable of achieving efficient production of an antimicrobial peptide in a microorganism, and a method for efficient mass production and separation of an antimicrobial peptide using the same. The gene construct of the present invention has a translationally coupled configuration of two independent and separate cistrons which encode an acidic peptide and a basic antimicrobial peptide, each having an opposite charge, under the control of a single promoter. The translationally coupled acidic peptide and basic antimicrobial peptide undergo charge-charge interaction simultaneously with expression thereof to neutralize the potential cytotoxicity of the antimicrobial peptide, resulting in prevention of antimicrobial peptide-mediated killing of host microorganisms. In addition, a conjugate of the acidic peptide and the antimicrobial peptide can be separated without chemical or enzymatic treatment. Therefore, it is possible to achieve easy mass production of antimicrobial peptides from recombinant microorganisms.

REFERENCES:
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patent: 10-0314721 (2001-01-01), None
Jang et al., “Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System”, Appl. Environmen. Microbiol. 75:3980-3986, 2009.
Lee et al. “Enhanced expression of tandem multimers of the antimicrobial peptide buforin II inEscherichia coliby the DEAD-box protein and trxB mutant”, Appl. Microbiol. Biotechnol. 58:790-796, 2002.
Park et al., “Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II”, Proc. Natl. Acad. Sci. 97:8245-8250, 2000.
Lee et al. “Acidic Peptide-Mediated Expression of the Antimicrobial Peptide Buforin II as Tandem Repeats inEscherichia coli”, Prot. Exp. Purif. 12:53-60, 1998.
Saito et al., “Direct Expression of a Synthetic Somatomedin C Gene inEscherichia coliby Use of a Two-Cistron System”, J. Biochem. 101:1281-1288, 1987.
Smith et al., “Generating a synthetic genome by whole genome assembly: øX174 bacteriophage from synthetic oligonucleotides” PNAS, Dec. 23, 2003, vol. 100, No. 26, pp. 15440-15445.

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