Efficient expression of sortase A from Staphylococcus aureus in Escherichia coli and its enzymatic characterizations
© The Author(s) 2017
Received: 5 December 2016
Accepted: 7 February 2017
Published: 18 February 2017
Sortase A (SrtA) is a transpeptidase found in Staphylococcus aureus, which is widely used in site-specific protein modification. However, SrtA was expressed in Escherichia coli (E. coli) in rather low level (ranging from several milligrams to 76.9 mg/L at most). The present study aims to optimize fermentation conditions for improving SrtA expression in E. coli.
Under the optimized media (0.48 g/L glycerol, 1.37 g/L tryptone, 0.51 g/L yeast extract, MOPS 0.5 g/L, PBS buffer 180 mL/L) and condition (30 °C for 8 h) in a 7-L fermentor, the enzyme activity and the yield of SrtA reached 2458.4 ± 115.9 U/mg DCW and 232.4 ± 21.1 mg/L, respectively, which were higher by 5.8- and 4.5-folds compared with initial conditions, respectively. The yield of SrtA also represented threefold increase than the previously reported maximal level. In addition, the enzymatic characterizations of SrtA (optimal temperature, optimal pH, the influence of metal irons, and tolerance to water-soluble organic solvents) were determined.
Enhanced expression of SrtA was achieved by optimization of medium and condition. This result will have potential application for production levels of SrtA on an industry scale. Moreover, the detailed enzymatic characterizations of SrtA were examined, which will provide a useful guide for its future application.
Sortase A (SrtA, EC number: 18.104.22.168) is a membrane-anchored transpeptidase first found in Staphylococcus aureus, which anchors surface proteins to cell wall by a cell-wall sorting reaction (Mazmanian et al. 1999). It recognizes a specific sorting signal peptide (Leu-Pro-X-Thr-Gly, with X standing for any amino acid except cysteine) at the C-terminal of target proteins, and cleaves the amide bond between threonine and glycine to generate a thioester intermediate; then the intermediate reacts with the amino group of pentaglycine cross-bridges resulting in the attachment of proteins to cell surface (Perry et al. 2002a, b). SrtA is a promiscuous enzyme, which can accept a variety of oligoglycine-modified nucleophiles as its substrate (Bentley et al. 2008). In addition, the truncated SrtA (Δ59 SrtA), by removing its original N-terminal transmembrane domain, showed good water solubility and retained the same transpeptidation activity as full-length SrtA (Ilangovan et al. 2001). Therefore, in recent years, SrtA-mediated ligation (SML) was widely used in protein-to-protein fusions (Witte et al. 2012), peptide and protein cyclizations (Wu et al. 2011), immobilization of biocatalyst onto solid surface (Chan et al. 2007), preparation of complex glycoconjugates (Guo et al. 2009), antibody–drug conjugation (Beerli et al. 2015,Voloshchuk et al. 2015), and in vivo protein modification (Glasgow et al. 2016).
With the development of SML, the demand for high-level expression of SrtA is very high. E. coli are the most studied expression system for SrtA production because of its effective genetic manipulation and cheap culturing cost (Vincentelli and Romier 2013). Since the recombinant Δ59 SrtA was first achieved in 1999 using pQE30 (Qiagen) as vector in E. coli (Ton-That et al. 1999), several groups have cloned and expressed SrtA using several commercial plasmids, including pET23b (Novagen) (Guo et al. 2009), pTWIN1 (New England Biolabs) (Bentley et al. 2008) and pBAD (Invitrogen) (Kim et al. 2002), etc., with slightly modified protocols. However, the reported yield of SrtA varied differently and remained at low level ranging from several milligrams to the maximum of 76.9 mg/L (Kruger et al. 2004). In our ongoing studies on SML for the synthesis of complex glycoconjugates (Wu et al. 2010, 2013), we realized that the low-level production of SrtA is difficult to meet the requirement of future industrial applications. Therefore, more endeavors should be made to improve the expression level of this important enzyme.
Heterologous expression of recombinant protein in E. coli is influenced by many factors, including medium composition, induction temperature, initial pH, and so on (Lee et al. 1997). In this study, Δ59 SrtA was cloned and successfully expressed in E. coli at first. Then, the optimization of fermentation conditions for SrtA production was manipulated by the combination of traditional one-factor-at-a-time approach and response surface methodology (RSM) at shake-flask stage, which is an effective and simplified method that has gained great success in the production of recombinant proteins (Papaneophytou and Kontopidis 2014). Finally, high-level expression of Δ59 SrtA was achieved in a 7-L fermentor, and the enzymatic characterizations of SrtA were examined.
Strain, plasmid, and media
The host strain E. coli BL21 (DE3) and plasmid pET28a were purchased from Novagen (Madison, WI). Luria broth (LB) medium (Tryptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L), Terrific broth (TB) medium (Tryptone 12 g/L, Yeast extract 24 g/L, Glycerol 4 g/L) with 100 mL/L PBS buffer (K2HPO4·3H20 164.3 g/L, KH2PO4 23.1 g/L), Super broth (SB) medium (Tryptone 30 g/L, Yeast extract 20 g/L, 3-(N-Morpholino) propanesulfonic acid (MOPS) 10 g/L), Soybean–peptone–yeast extract broth (SOB) medium (Tryptone 20 g/L, Yeast extract 5 g/L, NaCl 0.5 g/L, KCl 0.2 g/L) and 2× Yeast extract/tryptone (2× YT) medium (Tryptone 16 g/L, Yeast extract 10 g/L, NaCl 5 g/L), were used to perform the expression of SrtA, respectively.
Expression of SrtA in E. coli BL21 (DE3)
The genome of S. aureus was extracted by Genomic Extraction Kit (Qiagen, Valencia, CA, USA) and applied as the template for amplifying Δ59-srtA with the primer pairs: CAT GCC ATG GAA GCT AAA CCT CAA ATT CCG; and CGC GGA TCC TTA GTG GTG GTG ATG ATG ATG TTT GAC TTC TGT AGC TAC AAA GAT. The gel-purified PCR-amplified Δ59-srtA fragments were digested and inserted into the NcoI/BamHI site of pET28a. The plasmid (pET28a-Δ59-srtA) confirmed by DNA sequencing was transformed into E. coli BL21 (DE3) strain. The positive clones were propagated in LB medium at 37 °C overnight with constant shaking at 200 rpm. The seed culture (2%) was inoculated into 25 mL fermentation medium at 37 °C until the OD600 reached 0.6 and then incubated with 1 mM IPTG (isopropyl β-d-thiogalactoside) at 25 °C for 8 h. When the fermentation was completed, cells were pelletized and ultrasonicated by a probe VCX800 system (Sonics, Newtown, USA). The lysate supernatant were collected for the activity assay and purification.
SrtA activity assay
The specific substrate of SrtA (Dabcyl-QALPETGEE-Edans) was obtained from GL biochem Ltd. (Shanghai, China). The SrtA activity arrays were performed in 200-μL volume of 50 mM Tris–HCl buffer (including 150 mM NaCl, 30 mM CaCl2, 0.5 mg Dabcyl-QALPETGEE-Edans, pH 7.8), and 10 μL SrtA lysate supernatant. The reactions were carried out at 37 °C for 1 h by means of a Synergy H4 hybrid microplate reader (BioTek, Vermont, America), and the fluorescence intensity (FI) was detected with 350 nm for excitation and 495 nm for recordings. One unit of SrtA activity was defined as the amount of enzyme (mg) that was able to increase at the rate of one FI per minute in the 200-μL reaction mixture.
Purification of SrtA
The lysate supernatant and Ni–NTA agarose (Qiagen, Hilden, Germany) were loaded onto a gravity-flow column and incubated for 4 h at 4 °C. Then, the agarose was washed with a stepwise gradient of imidazole (10–40 mM) to eliminate intracellular contaminating proteins. The C-terminal His-tagged SrtA was eluted from the column using 500 mM imidazole and desalted using an Amicon Ultra 3 K device (Millipore, Billerica, USA). The concentration of purified SrtA was determined by the Bradford method and was used to calculate the yield of SrtA for each sample.
The optimal medium and several important fermentation conditions (induction time, induction temperature, and initial pH) for SrtA production were obtained by single factor optimization in 250-mL shaking flasks containing 25 mL of sterilized medium. The concentrations of alternative carbohydrates and nitrogenous compounds were equivalent to the concentrations of carbon and nitrogen resources in the initial medium (12 g/L tryptone; 4 g/L glycerol). In each experiment, one factor was changed, while the other factors were held constant. After fermentation, cell growth was monitored by measuring OD600 and correlated it with dry cell weight (DCW); the intracellular SrtA activity was measured by the method described in SrtA activity assay. Each experiment was performed in triplicate for the biological replicates, and the average values of enzymatic activity and biomass were used to select the optimal medium and conditions.
The Plackett–Burman design and effects of nine factors for SrtA production
High level (+1)
Low level (−1)
Yeast extract (g/L)
Induction occasion (OD600)
Loading volume (%)
Box and Behnken Design can propose three level designs for fitting response surfaces to get the best values for different variables by second-order polynomial model. Therefore, the concentration of three significant factors (glycerol, tryptone, and yeast extract) in the medium was optimized by the Box–Behnken Design tool of Design Expert 7.0 software. Each factor was coded with three levels (Additional file 1: Table S1) and seventeen experiments were performed in shaking-flask fermentation under previous fixed conditions (all cultivations were carried out in 250 mL shake flasks at 200 rpm). All experiments were performed in triplicates, and the average values of enzymatic activity and biomass were used to select the optimal medium and conditions. The P values were calculated by Duncan’s multiple range tests. The optimal concentrations of glycerol, tryptone, and yeast extract were determined by the prediction tool of software.
The enzymatic characterizations of SrtA
The effect of pH on SrtA activity was measured in the 200 μL reaction system at 37 °C (150 mM NaCl, 10 mM CaCl2, 0.5 mg Dabcyl-QALPETGEE-Edans) in a pH range of 3.0–11.0, using the appropriate buffers at concentration of 50 mM (3.0–5.0, sodium citrate; 6.0–8.0, sodium phosphate; 8.0–9.0, Tris–HCl; 10.0-11.0, and NaHCO3–NaOH). The optimal temperature for SrtA activity was determined at various temperatures ranging from 20 to 80 °C. To determine the effect of metal ions on SrtA activity, the enzyme assays were measured without additional metal ion (control) or with 5 mM different metal ions (Ca2+, Mn2+, Mg2+, Co2+, Cu2+, Fe2+, Ni2+ and Zn2+). As for the optimal concentration of Ca2+, the SrtA activities were detected at a concentration ranging from 1 mM to 100 mM. At last, the effect of soluble organic solvents (acetone, methanol, ethanol, acetonitrile, and dimethyl sulfoxide) on SrtA activity were determined, the enzyme assays were measured at a content of ranging from 10 to 50%. All experiments were performed in triplicate, and the mean values were used for calculations.
Results and discussion
Selection of the components of fermentation medium by single factor optimization
Because both carbon and nitrogen sources are important nutrients in the media for protein expression (Scott et al. 2002), these two nutrients were screened to find the optimal alternatives for SrtA expression. Five different types of carbohydrates (glucose, fructose, galactose, maltose, lactose, sucrose, dextrin, and starch) and nine nitrogenous compounds (beef extract, soya peptone, fishmeal, casein, urea, ammonium chloride, ammonium sulfate, and ammonium citrate) were used to replace the primary carbon and nitrogen sources in TB medium. As shown in Fig. 1c, glycerol was the suitable carbon source for SrtA expression which showed the best strain growth and enzymatic activity (1446.7 U/mg DCW). As for the nitrogen source, organic nitrogen source was superior to inorganic nitrogen source. As Fig. 1d shows, tryptone was the most suitable nitrogen source for SrtA expression, which gave the highest values of cell biomass and enzymatic activity (1407.1 U/mg DCW). Hence, glycerol and tryptone were selected in the subsequent experiments.
Influences of induction time, induction temperature, and initial pH on SrtA production
Determination of significant factors for SrtA production by Plackett–Burman design
The coefficient of determination (R 2 = 0.9884) indicates that 98.84% of model terms satisfy the model, and the fitting degree of the regression equation is good. The results revealed that the concentration of glycerol, tryptone, yeast extract are the significant factors (P value <0.05) and the other six factors have weaker effect (P value >0.05). Forecasted by Design-Expert software, the value of these six nonsignificant factors are defined as IPTG 1.5 mM, MOPS 0.5 g/L, PBS buffer 180 mL/L, induction occasion (OD600) 1.0, inoculum 2%, and loading volume 10%. Furthermore, the negative coefficients of glycerol (−162.08), tryptone (−243.91), and yeast extract (−201.77) indicated that the low concentrations of glycerol, tryptone, and yeast extract were beneficial for SrtA production.
Box–Behnken design results for the significant factors
The Box–Behnken design matrix and experimental results
SrtA production (U/mg DCW)
The enzyme activity and yield of SrtA performed in the optimized conditions (optimized medium, 30 °C for 8 h) and initial conditions (LB medium, 37 °C for 4 h) with shaking-flask fermentation and at 7-L fermentor level
7-L fermentor level
Activity (U/mg DCW)
Activity (U/mg DCW)
855.2 ± 95.1
44.2 ± 2.1
423.9 ± 8.8
52.1 ± 3.9
1732.3 ± 122.4
188.1 ± 18.3
2458.4 ± 115.9
232.4 ± 21.1
The enzymatic characterizations of Srt A
In summary, high-level expression of enzyme SrtA was achieved by a combination of single-factor optimization and response surface methodology in this study. Applying optimized medium (0.48 g/L glycerol, 1.37 g/L tryptone, 0.51 g/L yeast extract, MOPS 0.5 g/L, and PBS buffer 180 mL/L) and conditions (30 °C for 8 h), the highest enzyme activity and yield of SrtA could reach up to 2458.4 ± 115.9 U/mg DCW and 232.4 ± 21.1 mg/L, respectively. This formulation will have potential application of production levels of SrtA on an industry scale. In addition, the detailed enzymatic characterizations of SrtA were examined, which will provide a useful guide for its future application.
ZW and XZ deigned the experiment and analyzed the data, HH and XW performed the experiments. All the authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval, consent to participate, and consent for publication
All authors have read and approved to submit it to Bioresources and Bioprocessing. There is no conflict of interest of any author in relation to the submission.
This work was supported by the National Natural Science Foundation of China (21472070), the Project for Jiangsu Scientific and Technological Innovation Team, the Fund for Jiangsu Distinguished Professorship Program, and the State Key Laboratory of Natural and Biomimetic Drugs (K20140216), The Project was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,the 111 Project (No. 111-2-06), and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program.
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