Heterologous expression and purification of active divercin V41, a class IIa bacteriocin encoded by a synthetic gene in Escherichia coli

Abstract

Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41. To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain. The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form. The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 microg per 20 ml of culture). This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E. coli cytoplasm.

FIG. 1.

Construction of optimized divercin V41 gene in E. coli. (A) Amino acid sequence (sections 1 and 4), DNA sequence (sections 2 and 5), and frequency in E. coli K-12 of each noncoding codon on the x axis (sections 3 and 6) of wild-type (sections 1 to 3) and recombinant (sections 4 to 6) divercin V41. (B) Design, assembly, and construction of optimized divercin V41. The sequences of the sense and antisense oligonucleotides used for this study are shown. The designed restriction sites are underlined. Start and stop codons are shown in bold.

FIG. 2.

Expression of fusion protein in E. coli. The figure shows a glycine-SDS-PAGE gel stained with Coomassie blue R-250 (A) and the results of an ELISA (B) for total cell proteins obtained from the E. coli Origami strain carrying plasmid pCR04 before induction (lane 1), after 1 h of induction (lane 2), after 2 h of induction (lane 3), after 3 h of induction (lane 4), and after 3 h without induction (lane 5); the E. coli Origami strain carrying plasmid pET-32b after 3 h of induction (lane 6); and an ultra-low-range molecular mass marker (Sigma) (lane 7).

FIG. 3.

Purification process by affinity chromatography of recombinant divercin V41. (A) Tricine-SDS-PAGE gel stained with AgNO₃ showing a CSF of E. coli Origami (DE3)(pLysS) carrying plasmid pCR03 after 3 h of induction (lane 1), the first desalted IMAC-immobilized eluted fraction (lane 2), enterokinase cleavage products (lane 3), purified divercin RV41 (lane 4), a second IMAC-immobilized eluted fraction (lane 5), and an ultra-low-range marker (lane M). (B) Western blot of Tricine-SDS-PAGE products of CSF of E. coli Origami (DE3)(pLysS) carrying plasmid pET-32b after 3 h of induction (lane 1), a CSF of E. coli Origami carrying plasmid pCR03 after 3 h of induction (lane 2), the first desalted IMAC-immobilized eluted fraction (lane 3), enterokinase cleavage products (lane 4), purified divercin RV41 (lane 5), a second IMAC-immobilized eluted fraction (lane 6), and the supernatant of C. divergens V41 (lane 7). (C) Agar diffusion test. The CSF of E. coli Origami (DE3)(pLysS/pCR03) after 3 h of induction (spot 1), the first desalted IMAC-immobilized eluted fraction (spot 2), enterokinase cleavage products (spot 3), purified divercin RV41 (spot 4), a second IMAC-immobilized eluted fraction (spot 5), the supernatant of C. divergens V41 (spot 6), and the CSF of E. coli Origami carrying pET-32b after 3 h of induction (spot 7) are shown.

FIG. 4.

RP-HPLC elution profiles (C₁₈ nucleosyl) of buffer (profile 1), DvnRV41 purified after E. coli expression (profile 2), and DvnV41 purified from C. divergens V41 (profile 3). The retention time (in minutes) is given at the top of each elution peak.