Optimization of bacteriocin release protein (BRP)-mediated protein release by Escherichia coli: random mutagenesis of the pCloDF13-derived BRP gene to uncouple lethality and quasi-lysis from protein release

Abstract

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein beta-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.

FIG. 1

Template DNA and oligonucleotides used for random saturation mutagenesis of the mature part of the Lpp-BRP encoded by pJL17lpp. The region which was subjected to mutagenesis is translated, and the amino acids are shown between the coding (upper) and noncoding (lower) strands. The regions complementary to the sequences of the doped oligonucleotides (primers 2 through 5) and the flanking primers (primers 1 and 6) are indicated. Mutations which restored the carboxy-terminal epitope and mutations which introduced a HindIII site are indicated by asterisks. For specifications of the primers see Table 1.

FIG. 2

Schematic representation of the seven successive PCR experiments which were carried out to mutate the region coding for mBRP. Plasmid pJL17lpp was used as the template DNA for the subsequent PCRs performed with four doped oligonucleotides (primers 2 through 5) and two flanking oligonucleotides (primers 1 and 6). The product from PCR experiment G contains the region coding for the mature part of the mutated Lpp-BRP, flanked by SphI and HindIII sites.

FIG. 3

Immunoblot analysis of the release of β-lactamase (Bla) by E. coli C600 cells harboring a mutated BRP-encoding plasmid (A13, C09, C16, or C25). Cells were induced for production of the Lpp-BRP with 0.1 mM IPTG for 3 h, and then cells and the supernatant fraction (medium) were separated by centrifugation and equivalent amounts (0.2 optical density at 660 nm unit) were analyzed. Lane 1, A13 (wild type); lane 2, C09 (C17 and C18 gave the same results as C09); lane 3, C16; lane 4, C25. In some samples the β-lactamase appeared as two bands; these bands represent two different conformations of β-lactamase, a phenomenon which was caused by heating in SDS sample buffer.

FIG. 4

Growth, growth inhibition, and quasi-lysis of E. coli C600 cells harboring a plasmid encoding the wild-type Lpp-BRP (plasmid A13) or a plasmid encoding a mutated BRP (C25, C09, C16, C20, or C03). Cells were cultured in medium lacking Mg²⁺ and induced for production of one of the BRP derivatives with 0.1 mM IPTG. Symbols: ★, A13 without IPTG; ▪, C25; □, C09; ▴, C16; •, C20; ▵, C03; ○, A13 induced with IPTG. A plus sign indicates that the preparation was induced with IPTG.

FIG. 5

Immunoblot analysis of BRP in induced cells and in isolated inner and outer membrane fractions. (A) Whole cells. The plasmids encoding BRP (A13) or a mutant derivative (C09, C16, or C25) are indicated above the lanes. C17 and C18 are identical to C09. Equivalent amounts of cells were electrophoresed in the lanes of the tricine gel 3 h after induction. (B) Inner membrane (im) and outer membrane (om) fractions. Membranes were separated, and fractions containing inner membranes, as well as fractions containing outer membranes, were pooled and analyzed. Similar amounts were loaded onto the gel.

FIG. 6

Immunoblot analysis of the release of periplasmic chaperone FaeE by cells expressing wild-type BRP (encoded by A13) or a mutant derivative (encoded by C09 or C25). Control cells contained no BRP-encoding plasmid. Samples of cells (lanes C) and supernatant fractions (lanes S) were analyzed 2, 4, and 6 h after induction.

FIG. 7

Release of cloacin DF13 by cells expressing wild-type BRP or a mutant derivative. Cells containing pJL25 (encoding cloacin DF13) and plasmids A13, C09, C16, and C25, encoding BRP targeted by the stable BRP signal sequence, were cultured and induced with 100 ng of mitomycin per ml of medium and 0.1 mM IPTG. Samples were taken 5 h after induction, cells and medium were separated by centrifugation, and the presence of cloacin DF13 in the supernatant fractions was determined by SDS-PAGE, followed by protein staining. Lane 1, cells (A13) (all other cell fractions produced a similar protein pattern); lane 2, molecular mass markers from New England Biolabs (molecular masses, from top to bottom, 212, 158, 116, 97.2, 66.4, 55.6, 42.7, 36.5, 26.6, and 20.0 kDa); lanes 3 through 6, supernatant fractions of cultures of cells expressing A13, C09, C16, and C25 BRP, respectively. Equivalent amounts of the supernatant fractions were electrophoresed on the gel. The position of cloacin DF13 (Clo) (about 66 kDa) is indicated.