Coexpressing the Signal Peptide of Vip3A and the Trigger Factor of Bacillus thuringiensis Enhances the Production Yield and Solubility of eGFP in Escherichia coli

Abstract

Many fusion tags have been developed to improve the expression of recombinant proteins. Besides the translocation of cargo proteins, the signal peptides (SPs) of some secretory proteins, such as the ssTorA and Iasp, have been used as an inclusion body tag (IB-tag) or the recombinant expression enhancer in the cytosol of E. coli. In this study, the approach to utilize the SP of Vip3A (Vasp) from Bacillus thuringiensis (Bt) as a fusion tag was investigated. The results showed that either the Vasp or its predicted N- (VN), H- (VH), and C-regions (VC), as well as their combinations (VNH, VNC, and VHC), were able to significantly enhance the production yield of eGFP. However, the hydrophobic region of the Vasp (VH and/or VC) made more than half of the eGFP molecules aggregated (VeGFP, VHeGFP, VCeGFP, VNHeGFP, VNCeGFP, and VHCeGFP). Interestingly, the addition of the Bt trigger factor (BtTF) led to the neutralization of the negative impact and solubilization of the fusion proteins. Therefore, the coexpression of Vasp or its derivates with the chaperone BtTF could be a novel dual-enhancement system for the production yield and solubility of recombinant proteins. Notably, EcTF was unable to impact the solubility of Vasp or its derivates guided proteins, suggesting its different specificities on the recognition or interaction. Additionally, this study also suggested that the translocation of Vip3 in the host cell would be regulated by the BtTF-involved model.

Keywords: Bacillus thuringiensis; Bt trigger factor; Vip3; eGFP; fusion tag; signal peptide.

Figure 1

The alignment of the representative Vsp sequences. The predicted N-, H-, and C-regions of SP are indicated. The numbers of genes sharing the corresponding sequences are indicated in parentheses.

Figure 2

The hydrophobicity scale of SPs of Vip3A (A), MBP (B), pelB (C), and TorA (D) proteins.

Figure 3

The expression of VeGFP in the E. coli MC4100 strain controlled by the constitutive promoter P_ac. The samples of eGFP (27.9 kDa), VeGFP (lane 2, 31.8 kDa), and IeGFP (lane 3, 33.1 kDa) were prepared at 8 (A) and 12 h (B) after inoculation, respectively. Lane “–” is the negative control prepared from M304 cells. Lane “M” is the molecular weight standard. The arrows indicate the target bands of the recombinant proteins. The target proteins were also verified by western blot (C).

Figure 4

Coexpression analysis of VeGFP with several chaperones. For each sample, the total proteins (T), the soluble component after cell lysis (S), and the precipitates (P) were loaded, respectively. The black arrows indicate the corresponding chaperones (EcSecB, 17.3 kDa; SecB7577, 17.3 kDa; SecB142, 15.9 kDa; SecB142-7577, 15.9 kDa; BtCsaA, 11.9 kDa; BtSecA, 95.0 kDa; BtTF, 47.3 kDa). The hollowed arrows indicate the target recombinant proteins.

Figure 5

The fluorescent signal distribution of VeGFP and VNeGFP in E. coli BL21-star (DE3) cells. The corresponding cells expressing VeGFP (a–c) or VNeGFP (g–h) individually, or with BtTF d–f for VeGFP and j–l for VNeGFP) were observed using an inverted confocal microscope (Leica SP8). For each sample, the image in the third row was merged with the corresponding bright-field image in the first row and the fluorescent one in the second row. The scale bar represents 10 μm.

Figure 6

Diagram of Vasp variants (A) and their effect (B) on the expression of eGFP. In panel B, the target bands of eGFP (lane 1), VeGFP (lane 2), VNeGFP (lane 3, 29.2 kDa), VHeGFP (lane 4, 29.1 kDa), VCeGFP (lane 5, 29.5 kDa), VNHeGFP (lane 6, 30.3 kDa), VNCeGFP (lane 7, 30.7 kDa), and VHCeGFP (lane 8, 30.6 kDa) are indicated by hollowed arrows, respectively. Lane “–” is the negative control prepared from BL28aD cells. Lane “M” is the molecular weight standard.

Figure 7

The solubility analysis of the fusion fluorescent proteins with (A) or without (B) the coexpression of BtTF. For each sample, the total proteins (T), the soluble component after cell lysis (S), and the precipitates (P) were loaded. The black arrows indicate the target bands of BtTF and the hollowed arrows indicate the target recombinant proteins including eGFP, VeGFP (V), VNeGFP (VN), VHeGFP (VH), VCeGFP (VC), VNHeGFP (VNH), VNCeGFP (VNC), and VHCeGFP (VHC). Lane “M” is the molecular weight standard.

Figure 8

The effect of the Vasp derivates and BtTF on the expression of GDF8 protein. (A) The diagram of expression cassettes of VNhteGDF8, VNHhteGDF8, and V3NhteGDF8. The expression of VNhteGDF8 (B, 16.9 kDa), V3NhteGDF8 (C, 19.2 kDa), and VNHhteGDF8 (D, 18.0 kDa) was analyzed by SDS-PAGE. For each sample, the total proteins (T), the soluble component after cell lysis (S), and the precipitates (P) were loaded. Lane “M” is the molecular weight standard. The black arrow indicates the BtTF and the hollowed arrow indicates the recombinant proteins.