Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli

Abstract

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA(218)) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 x 10(4) E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23 degrees C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA(218) fusion protein at 23 and 30 degrees C but unexpectedly not at 37 degrees C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA(218) was detected only in the L448F mutant (AE104F) at 23 degrees C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA(218) fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37 degrees C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.

FIG. 1.

Structure of hlyB and hlyD genes on pSTV-HlyBD as a target for mutagenesis. Primers for random mutagenesis, including restriction enzyme recognition sites, are indicated by arrows. The ATPase region of HlyB is shown by a gray box.

FIG. 2.

Proteolytic degradation of skim milk by secretion of subtilisin E-HlyA₂₁₈. E. coli JM109 strains carrying pSub-HlyA₂₁₈ with pSTV-HlyBD (wild type [WT]), pSTV28 (negative control [NC]), pAE104 (AE104), and pAE129 (AE129) were selected and grown at 23°C on an LB agar plate containing 2% skim milk, 0.4 mM IPTG, 100 μg of ampicillin/ml, and 30 μg of chloramphenicol/ml.

FIG. 3.

Relative secretion levels of subtilisin E-HlyA₂₁₈ in HlyB and HlyD mutants. Activities of subtilisin E in the culture supernatants were measured 24 h after induction with IPTG at 23°C. Each bar represents the mean ± standard deviation (n = 3). WT, wild type.

FIG. 4.

Secretion activities of HSA14-1 scFv-HlyA₂₁₈ in HlyB and HlyD mutants. The amount of HSA14-1 scFv-HlyA₂₁₈ in the culture supernatant was measured by ELISA. HSA14-1 scFv-HlyA₂₁₈ expression was induced by addition of 0.4 mM IPTG at 37°C for 5 h (white bars), at 30°C for 5 h (gray bars), and at 23°C for 24 h (black bars). Secretion efficiency is shown as micrograms of secreted protein per liter per OD₆₆₀ unit. WT, wild type.

FIG. 5.

Specific secretion of HSA14-1 scFv-HlyA₂₁₈. After IPTG induction at 23°C for 24 h, fractions of periplasm, spheroplast, and culture supernatants of equal amounts of cells (0.05 OD₆₆₀ equivalents of periplasm and spheroplast samples and 0.02 OD₆₆₀ equivalents of supernatants) were detected with anti-σ⁷⁰ MAb (row 1), anti-E tag MAb (row 2), and anti-β-lactamase MAb (row 3) by Western blotting. Anti-mouse immunoglobulin G-AP was used as a secondary antibody. NC, negative control; WT, wild type.

FIG. 6.

Mutation sites in HlyB and HlyD mutants. The ATP-binding domain or NBD in HlyB is shown by a gray box. Putative TMDs in HlyB and HlyD are shown by stippled boxes. The conserved switch II region is shown by a hatched box. The mutation site in pAE129 (G654S) is shown by an asterisk.

FIG. 7.

Secretion activities of HSA14-1 scFv-HlyA₂₁₈ in AE104-based mutants. E. coli JM109 strains carrying pHSA14-1-HlyA₂₁₈ with pSTV-HlyBD (WT), pAE104 (AE104), pAE104A (AE104A), pAE104B (AE104B), pAE104C (AE104C), pAE104D (AE104D), or pAE104E (AE104E) were cultured, and protein expression was induced by IPTG at 23°C for 24 h. The level of secreted HSA14-1 scFv was measured by ELISA. Secretion activities were calculated relative to that of AE104 (100%).

FIG. 8.

Comparison of secretion activities of the wild type (WT), AE104, and AE104F. E. coli JM109 strains carrying pHSA14-1-HlyA₂₁₈ with pSTV-HlyBD (WT), pAE104 (AE104), or pAE104F (AE104F) were cultured, and protein expression was induced by IPTG at 23°C for 24 h. The level of secreted HSA14-1 scFv was measured by ELISA. Secretion activities were calculated relative to that of AE104 (100%).

FIG. 9.

Secretion of intracellular proteins by mutant HlyB and HlyD strains. (A) Secretion of c-Myc-HlyA₂₁₈ was evaluated by using E. coli cultures carrying pMyc-HlyA₂₁₈ with pSTV-HlyBD (wild type [WT]) or pAE104F (AE104F) or carrying pSTV28 with pMyc-HlyA₂₁₈ (negative control [N]). (B) Secretion of PTEN-HlyA₂₁₈ was evaluated by using E. coli cultures carrying pPTEN-HlyA₂₁₈ with pSTV-HlyBD (WT) or pAE104F (AE104F) or carrying pSTV28 with pPTEN-HlyA₂₁₈. Culture media were taken at 1, 2, and 5 h after IPTG induction at 37°C and at 4, 8, and 24 h after IPTG induction at 23°C as indicated. Then, 0.004 OD₆₆₀ equivalent was applied to the gel, and the proteins were separated by SDS-PAGE. The secretion levels of target proteins were evaluated by Western blotting. Negative control cells were cultured for 24 h after induction at 23°C.