Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli

Abstract

Protein secretion in Escherichia coli is usually led by a signal peptide that targets the protein to specific secretory pathways. In this study, we demonstrated that the superfolder green fluorescent protein (sfGFP) could be served as a non-signal peptide to guide protein auto-secretion in E. coli. This auto-secretion was characterized as a three-step process through the sub-cellular localization analysis: inner membrane trans-location followed by anchoring at outer membrane, and then being released into culture media. We further determined that the beta-barrel structure and net negative charges of sfGFP played important roles in its auto-extracellular secretion property. Using sfGFP as a carrier, heterologous proteins ranging from peptide to complex protein, including antibacterial peptide PG4, endo-beta-N-acethylglucosamindase H (Endo H), human arginase-1 (ARG1), and glutamate decarboxylase (GAD) were all successfully expressed and secreted extracellularly when fused to the carboxyl end of sfGFP. Besides facilitating the extracellular secretion, sfGFP fusion proteins can also be correctly folded and formed the active complex protein structure, including the trimetric human ARG1 and homo-hexametric GAD. This is the first report that sfGFP can guide the secretion of recombinant proteins out of the cells from cytoplasm in E. coli without affecting their conformation and function.

Figure 1

Diagrammatic sketch. (a) Possible secretory mechanism ofthe sfGFP fusion protein; (b) Constructs generated in this study; T7: T7 promoter; His₆: 6 × His epitope tag; sfGFP: superfolder green fluorescent protein; scGFP: super charging green fluorescent protein; PG4: antibacterial peptide PG4; Endo H: endo-beta-N-acethylglucosamindase H; ARG1: human arginase-1; GAD: Glutamate decarboxylase; T7 ter: T7 terminator.

Figure 2

The characterization of sfGFP secretion in E. coli. (a) SDS-PAGE analysis the expression of sfGFP and scGFP in E. coli; 1: scGFP expressed in E. coli strain Rosetta Blue; 2: sfGFP expressed in E. coli strain Rosetta Blue; 3: negative control (plasmid pET23a) expressed in E. coli strain Rosetta Blue; (b) Western-Blot analysis of the expression of the sfGFP and scGFP in E. coli; the analysis was carried out using the anti-GFP monoclonal antibody; (c) Western-Blot analysis of the extracellualr secretion of sfGFP in E. coli; anti-GroEL:cytoplasmic protein GroEL monoclonal antibody; anti-beta lactamase: periplasmic protein beta lactamase monoclonal antibody; anti-GFP: GFP monoclonal antibody; (d) SDS-PAGE analysis of the sub-fraction of sfGFP in E. coli; (e) Western Blot analysis of the sub-fraction of sfGFP in E. coli; and the analysis was carried out using the periplasmic protein beta-lactamase monoclonal antibody; (f) Native-PAGE analysis of the sub-fractions of sfGFP in E. coli; (g) Western Blot analysis of sub-fractions of sfGFP in E. coli; anti-His: 6 × His monoclonal antibody. The lane markers were in Fig. 2, T: total protein; S: supernatant of cell culture media; C: cytoplasmic protein; P: periplasmic protein; OM: outer membrane protein.

Figure 3

Analysis the auto secetion mechanism of sfGFP in E. coli. (a) Western blot annalysis of the extracellular expression of sfGFPs in E. coli; N10: mutant of sfGFP with deletion of 10 amino acids at its N-terminus; N20: mutant of sfGFP with deletion of 20 amino acids at its N-terminus; anti-GFP: GFP monoclonal antibody; all samples were tested by using GFP monoclonal antibody; (b) Western blot annalysis of the extracellualr secretion of sfGFP-MPH and N20-MPH in E. coli; all samples were tested by using 6 × His mono-antibody; (c) Enzyme activity assay of extracellular secretion fusion protein sfGFP-MPH and N20-MPH; N20-MPH: N-terminus 20 amino acids of sfGFP fused to the N-terminal of MPH; sfGFP-MPH: sfGFP fused to the N-terminal of MPH; (d) SDS-PAGE annalysis of the sub-fraction of sfGFP-MPH in E. coli; (e) SDS-PAGE annalysis of the sub-fraction of mCherry-MPH in E. coli; (f) SDS-PAGE annalysis of the expression of sfGFP and scGFP in E. coli; (g) SDS-PAGE annalysis of the expression of sfGFP and its mutants in E. coli; sfGFP(−18) ~ scGFP: sfGFP mutants containing net charges; T: total cell protein; SP: soluable protein of the target peotein; S: supernatant of culture media; S1~S5: supernatant of culture medium after expression process and stayed at room temperature for 0~4 days; C: cytoplasmic protein; P: periplasmic protein; OM: outer membrane protein.

Figure 4

Analysis the auto secretion of sfGFP fusionsin E. coli. (a) Western Blot analysis of the extracellular expression of the sfGFP-PG4 in E. coli; C-T: total cell protein of the control; C-S: extracellular protein of the control; S1~S5: after expression, supernatant protein sample of culture media stayed in room temperature for one to five days; (b) SDS-PAGE analysis of the sub-fraction of the sfGFP-PG4 in E. coli; (c) SDS-PAGE analysis of the sub-fraction of sfGFP-Endo H in E. coli; (d) Enzyme activity analysis of sfGFP-Endo H; 1: native protein Rnase B; 2: native protein Rnase B digested by the recombinant protein Endo H (New England Biolab); 3~5: native protein Rnase B digested by sfGFP-Endo H (dilution 1:100, 1:1000, 1:10000); (e) SDS-PAGE analysis of the sub-fraction of sfGFP-ARG1 in E. coli; (f) Exclusion chromatography and SDS-PAGE analysis of sfGFP-ARG1; Lane 1: un-denatured sample of fusion protein sfGFP-ARG1; Lane 2: denatured sample of sfGFP; (g) SDS-PAGE analysis of the sub-fraction of sfGFP-GAD in E. coli; T: total cell proteins; S: supernatant of culture media; C: cytoplasmic protein; P: periplasmic protein; OM: outer membrane protein.

Figure 5

Optimazation of the extracellular secretion of sfGFP-MPH in E. coli. Blueline stands for theconcetration of extracellular secretion sfGFP-MPH;Green linestands for the special activity of extracellular secretion sfGFP-MPH; and Red line stands for the fluorescence intensity of extracellular secretion sfGFP-MPH.

Figure 6

Analysis the extracellular secretion of sfGFP-ARG1 in fed-batch fermentation condition. (a) Cells concentration in fed-batch fermentation condition; (b) Fluorescence intensity of extracellar secretion sfGFP-ARG1 in fed-batch fermentation condition; (c) Protein concentration of extracellar secretion sfGFP-ARG1 in fed-batch fermentation condition; (d) Enzyme activity of extracellar secretion sfGFP-ARG1 in fed-batch fermentation condition.