Abstract
In prokaryotes, the twin-arginine translocation (Tat) signal peptide is specific for the secretion of folded proteins. On the other hand, the enhanced green fluorescent protein (EGFP) is widely used as a reporter protein in biological studies. In this work, the plasmids pBT2-ET-EGFP (encoding EGFP gene without signal peptide) and the plasmid pBT2-ETG (encoding the Tat-EGFP fusion protein with a staphylococcal TAT signal peptide) were respectively employed to investigate the translocation effect of Tat-EGFP by Tat signal peptide from the cytoplasm to periplasm of E. coli host. By examinations with microscopic fluorescence, SDS-PAGE and Western blot, as expected, the Tat-EGFP was successfully translocated into the periplasm of the E. coli host cells, but EGFP without signal peptide not. Furthermore, both kinds of EGFPs were not secreted into the fermentation broth. Therefore, this result primarily revealed the insight into the role of the heterologous signal peptide for the transmembrane translocation in E. coli.
Keywords
1 Introduction
A variety of protein transport systems have been found in bacteria for decades. Based on their structure, the signal peptide can be mainly divided into the following kinds: Sec (Secretion Translocation Pathway), Tat (Twin Arginine Translocation) [1], LIP (Lipoprotein), Com (type IV pili structure), and ATP-binding cassette (ABC) signal peptide [2]. To date, Sec pathway is widely used for exogenous protein secretion in genetic engineering, but Sec transport system can only secrete unfolded or partially folded proteins [3]. However, Tat signal peptide secretion system can secrete the correctly-folded protein to the outside of the cell [4, 5]. Tat pathway was first found in E. coli. Bolhuis et al. found that two integral cytoplasmic membrane proteins, TatB and TatC, consist a structural and functional unit of the twin-arginine translocases in E. coli. In the field of genetic engineering, it can be used to secrete heterologous proteins that cannot be transported by Sec translocation pathway [6]. Currently, it has become a focus of protein transportation research in the world due to its feature to help translocate fully folded proteins across the bacterial plasma membrane [7].
Green fluorescent protein (GFP) can be well expressed as a reporter protein in a large number of prokaryotic [8] and eukaryotic cells without affect by the biological, tissue or genotype [9]. In addition, GFP possesses a small molecular weight (ca. 27 kDa), and does not affect the property and function of other fused proteins [4]. Thus, the target protein can be fused with GFP for the localization analysis [10]. Moreover, its expression has the advantages of easy detection, high sensitivity, stable fluorescence, non-toxic to cells, among others. Therefore, GFP can be directly used for the determination of living cells characteristics, and is widely used in various research fields of the life sciences [11].
In this study, the enhanced green fluorescent protein (EGFP) was used as a reporter protein to study the protein secretion properties by Tat pathway in E. coli [12]. A Tat signal peptide from Staphylococcus carnosus TM300 [7, 13, 14] was ligated with EGFP gene to construct the Tat-EGFP fusion gene. The EGFP and fused Tat-EGFP (EGFPs) were employed to investigate its role in EGFP translocation in E. coli host.
2 Materials and Methods
2.1 Strains and Plasmids
E. coli DH5α was obtained from the preservation of Tianjin Municipal Industrial Microbiology Key Laboratory, Tianjin, China. Staphylococcus carnosus TM300 and plasmid pBT2 (Cmr, Ampr) were kindly offered by Professor Dr. Friedrich Goetz of the Department of Microbial Genetics, Eberhard-Karls-Universitaet, Tuebingen, Baden-Wuerttemberg, Germany. The plasmids pBT2-ETG encoding the Tat-EGFP fusion protein with a Tat signal peptide of iron-dependent peroxidase EfeB (GenBank ID: CAL29117.1) in S. carnosus TM300 and pBT2-ET-EGFP only encoding EGFP gene without signal peptide were constructed in our previous work [12,13,14].
2.2 Media and Culture Condition
LB broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) was employed for the cultivation of E. coli. The solid LB medium was prepared by the addition of 20 g/L agar. The ampicillin resistance was used for the screening of the recombinants at final concentration of 100 μg/mL.
2.3 Enzymes and Reagents
The NheI and BamHI restriction enzymes, T4 DNA ligase and low molecular weight proteins marker were purchased from Fermentas Inc., (Burlington, Ontario, Canada). 2× Taq PCR Mastermix, plasmid extraction kit, 1 kb DNA Ladder and DNA Marker III were bought from Tiangen Biotech (Beijing) Co., Ltd., (Beijing, China). Lysozyme (20,000 U/mL), EGFP antibody, PVDF film and Color Developing Reagent (DAB) kit were bought from Beyotime Biotechnology Research Institute (Nantong, Jiangsu, China). Cell lysis buffer was composed of 0.8 mg/mL lysozyme, 20% sucrose, 50 mmol/L Tris and 1 mmol/L EDTA (pH 8.0). All other reagents were analytical or biological grade.
2.4 Extraction of EGFPs from E. coli
For the extraction of EGFPs (EGFP and Tat-EGFP), single recombinant colonies of E. coli DH5α/pBT2-ET-EGFP and E. coli DH5α/pBT2-ETG were respectively grown in 50 mL LB broth at 180 r/min and 37 °C for 16 h. The cell pellets were harvested by centrifugation at 12,000 r/min and 4 °C for 15 min. Proteins in the culture broth were treated with 10% trichloroacetic acid (TCA) for 12 h at 4 °C. The cells were resuspended in 2 mL PBS buffer, disrupted by sonication, and recovered the supernatant from cell fragments by centrifugation as the cytoplasm fraction.
The fermentation broth of E. coli was centrifuged at 12,000 r/min and 4 °C for 10 min, then the cell precipitate was washed twice with PBS (pH 7.4) for collection. Then the cell lysis buffer was added to fully suspend the bacterial cells on ice bath for 30 min. The supernatant after centrifuge was the periplasm protein fraction.
The culture broth of E. coli was centrifuged, and the supernatant was extracted and then mixed with 10% TCA precipitation, kept statically at 4 °C overnight. The precipitation was washed once with 100–80% acetone, and then the proteins in the fermentation broth was precipitated by centrifugation, and dried at room temperature as the fermentation broth fraction.
2.5 Detection of EGFP
The expression of the EGFPs in the cells and fermentation broth of E. coli DH5α/pBT2-ET-EGFP and E. coli DH5α/pBT2-ETG was observed by fluorescence microscopy [OLYMPUS (China) Co., Ltd., Beijing, China], and analyzed by SDS-PAGE and Western blot.
3 Result
3.1 Fluorescence Observation of EGFPs in E. coli DH5α Cells
By dropleting the E. coli transformant culture broth on the slide surface with cover glass, fluorescence was observed under a fluorescent microscope. In Fig. 1, E. coli DH5α/pBT2-ET-EGFP and E. coli DH5α/pBT2-ETG cells emitted green fluorescence, but the control E. coli DH5α not. The results indicated that both EGFPs genes were able to be expressed in E. coli DH5α host.
3.2 Fluorescence Spectrophotometer Detection
The detection of EGFP and Tat-EGFP in periplasm and fermentation broth of various E. coli hosts were performed using an fluorescence spectrophotometer (Hitachi F-7000, Japan). Only Tat-EGFP gave an obvious fluorescence emission peak at 510 nm in the periplasm of E. coli DH5α/pBT2-ETG, but not EGFP for E. coli DH5α/pBT2-ET-EGFP, indicating that the staphylococcal Tat signal peptide plays a positive role for transmembrane protein transport of Tat-EGFP (Fig. 2).
Additionally, no emission peak was found at 510 nm from the both fermentation broth, suggesting that both kinds of EGFPs could not be secreted into the fermentation broth from their E. coli host cells, respectively. This can be referred to the lack of translocation and the outer membrane block for EGFP as well as the later for Tat-EGFP.
3.3 SDS-PAGE Analysis
For the overnight cultures of E. coli DH5α, E. coli DH5α/pBT2, E. coli DH5α/pBT2-ET-EGFP and E. coli DH5α/pBT2-ETG, SDS-PAGE was used for the analysis of EGFPs in the cytoplasm, periplasm and fermentation broth to determine whether the EGFPs could be secreted into the culture broth (Fig. 3).
3.4 Western Blot Verification
In this work, whether the expressed EGFPs can be translocated into the periplasm and fermentation broth from various E. coli hosts were further detected by Western blot. The Western blot experiment here also exhibited the same result as SDS-PAGE, i.e. both EGFP and Tat-EGFP were correctly expressed in the cytoplasm of E. coli hosts, but only Tat-EGFP was expressed in the periplasm (Fig. 4), and none of them could be transported into the fermentation broth (data not shown).
4 Conclusion
To our best knowledge, this is the first report that the constructed Tat-EGFP was successfully translocated in the periplasm E. coli DH5ɑ host by a Tat signal peptide from S. carnosus TM300, but EGFP not. Our results revealed that the heterologous staphylococcal Tat signal peptide is recognized by the E. coli host, and exerts a positive role in the transmembrane secretion of Tat-EGFP, which sheds the light as a novel pathway for the secretion of the heterologous proteins in E. coli and probably other bacteria.
References
Lee PA, Tullman D, Georgiou G et al (2006) The bacterial twin-arginine translocation pathway. Annu Rev Microbiol 60:373–395
Bogsch EG, Sargent F, Stanley NR et al (1998) An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J Biol Chem 273(29):18003–18006
Jensen CL, Stephenson K, Jørgensen ST et al (2000) Cell associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system. Microbiology 146(10):2583–2594
Zhang G, Gurtu V, Kain SR (1996) An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochem Biophys Res Commun 227(3):707–711
Tjalsma H, Bolhuis A, Jongbloed JDH et al (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64(3):515–547
Thiemann V, Saake B, Vollstedt A et al (2006) Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii. Appl Microbiol Biotechnol 72(1):60–71
Biswas L, Biswas R, Nerz C et al (2009) Role of the twin arginine translocation pathway in Staphylococcus. J Bacteriol 191(19):5921–5929
Tünnemann G, Martin RM, Haupt S et al (2006) Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J 20(11):1775–1784
Sheen J, Hwang S, Niwa Y et al (1995) Green-fluorescent protein as a new vital marker in plant cells. Plant J 5(8):777–784
Chalfie M, Tu Y, Euskirchen G et al (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805
Chen Y, Müller JD, Ruan QQ et al (2002) Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy. Biophys J 82(1):133–144
Yu C, Zheng X, Zhu Y et al (2011) Construction of tat-gfp fusion gene and its expression in Staphylococcus carnosus. Biotechnol Bull 8:203–207
Xu B, Cheng Y, Wang L et al (2015) Construction of Eschericha coli-Staphylococcus shuttle vector for EGFP expression and potential secretion via Tat pathway. Lect Notes Electr Eng 333:171–180
Gao Q, Xu B, Cheng Y et al (2014) GFP translocation of twin-arginine secretion pathway in Staphylococcus carnosus. J Tianjin Univ Sci Technol 5:1–5
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (31370075, 31101275 and 61603273), the Undergraduate Laboratory Innovation Fund of Tianjin University of Science and Technology of China (1504A304X) and the Youth Innovation Fund of Tianjin University of Science and Technology of China (2014CXLG28).
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Zhou, QX., Zhang, J., Wang, MN., Yang, WH., Zhang, J., Gao, Q. (2018). Study on a Staphylococcal Tat Signal Peptide Guided EGFP Translocation in E. coli . In: Liu, H., Song, C., Ram, A. (eds) Advances in Applied Biotechnology. ICAB 2016. Lecture Notes in Electrical Engineering, vol 444. Springer, Singapore. https://doi.org/10.1007/978-981-10-4801-2_9
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