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Efficient extracellular expression of transpeptidase sortase A in Pichia pastoris
Protein Expression and Purification 133 (2017) 132e138
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Efficient extracellular expression of transpeptidase sortase A in Pichia pastoris Xinrui Zhao a, Haofei Hong a, Zhimeng Wu a, b, * a The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road, Wuxi 1800, China b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2016 Received in revised form 28 February 2017 Accepted 13 March 2017 Available online 15 March 2017
In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly3polystyrene beads. © 2017 Elsevier Inc. All rights reserved.
Keywords: Sortase A Extracellular expression Immobilization Optimization Pichia pastoris
1. Introduction Sortase A (SrtA, EC: 3.4.22.70) is a transpeptidase found in Staphylococcus aureus [1]. It anchors surface proteins with a specific C-terminal sorting signal (LPXTG, X stand for any amino acid except cysteine) to cell wall by cleaving the amide bond between threonine and glycine and transferring the thioester intermediate to the amino group of pentaglycine cross-bridges [2]. Due to its sitespecific transpeptidation reaction, SrtA has been widely used as a ligation tool in the fusion of protein domains with their preserved functionality [3], post-translational modification of bionanoparticles [4], peptides and protein cyclization [5], preparation of complex glycoconjugates [6] and live cell labelling [7]. Especially, SrtAmediated ligation was applied to synthesize antibody-drug conjugates as highly promising biotherapeutics [8]. Furthermore, efforts to evolve SrtA by site-mutations have generated variants of SrtA with improved kinetics, which were applied in antibody and protein conjugation reactions [9,10]. Thus, SrtA has great potential applications in biotechnology industry.
* Corresponding author. The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, China. E-mail address: [email protected] (Z. Wu). http://dx.doi.org/10.1016/j.pep.2017.03.010 1046-5928/© 2017 Elsevier Inc. All rights reserved.
So far, expression of SrtA has long been limited in intracellular expression in Escherichia coli by using various commercial plasmids, including pET23b (Novagen) [6], pQE30 (Qiagen) [11] and pBAD (Invitrogen) [12] etc.. Although these strategies could produce SrtA for the laboratory use, the expression level of SrtA maintained at low level with yields ranging from several milligrams to maximum 76.9 mg/L [13]. Disadvantages of these intracellular expression strategies, including cumbrous cell disruption and timeconsuming downstream purification process, obstructed its future industrial application. In addition, compared with the extensive applications of SrtA, few endeavors were made to improve or optimize the expression level of this important enzyme. Therefore, new strategy for the efficient extracellular expression of SrtA is highly demanded. In the last two decades, the methylotrophic Pichia pastoris has proven to be an excellent host for the production of a variety of heterologous proteins in both academia and industry. This expression system featured with ease genetic manipulation, high cell densities using inexpensive medium and post-translational modification. In addition, various P. pastoris hosts, such as GS115 (Mutþ phenotype), SMD1168 (Mutþ phenotype, defective in the vacuole peptidase A) and KM71 (Muts phenotype) etc. [14], and promoters (including the methanol-induced AOX1 promoter and constitutive GAP promoter etc.) [15] were available for screening the specific expression requirement. In particular, with the
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successful a-factor signal peptide from Saccharomyces cerevisiae [16], many large-scale extracellular production of recombinant proteins was achieved at high levels in P. pastoris [17]. This a-factor signal peptide can efficiently mediate the secretion of heterologous proteins through endoplasmic reticulum, Golgi apparatus and plasma membrane [18]. Furthermore, target protein was harvested from the culture medium directly without cell-disruption or refolding operation, which greatly simplify the downstream purification steps [19]. Therefore, the P. pastoris system has a potential for the efficient extracellular expression of SrtA. In this study, extracellular expression of a truncated version of SrtA (D59-SrtA with good solubility and the same transpeptidation activity with full-length SrtA) [20] in P. pastoris was investigated. By adopting various strategies (different hosts, promoters and gene copy numbers) for protein expression in P. pastoris, a series of recombinant engineered strains were constructed and the extracellular expression of SrtA was firstly achieved. Based on SDS-page analysis and activity assay, a suitable strain to secret expression of SrtA was obtained. Then, the expression level of SrtA was further enhanced by the optimization of fermentation conditions. Finally, the transpeptidation activity of secreted SrtA was confirmed by immobilization of enhanced green fluorescent protein (EGFP) onto glycine-modified polystyrene beads. 2. Materials and methods 2.1. Strain, plasmid and media The P. pastoris host strain GS115 (his4, Mutþ, His-, aox1þ, aox2þ), SMD1168 (his4, pep4, Mutþ, His-, aox1þ, aox2þ) and KM71 (his4, MutS, His-, aox1-, aox2þ) and plasmid pPIC9k were purchased from Invitrogen (Carlsbad, USA). The E. coli JM109 (Novagen, Madison, WI) was used as the host for cloning and DNA sequencing. E. coli strain was cultured in LB medium (Tryptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L) with 100 mg/mL ampicillin. P. pastoris strains were grown in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose), BMGY medium (13.4 g/L YNB without amino acids, 20 g/L peptone, 10 g/L yeast extract, 10 g/L glycerol and 100 mM potassium phosphate) or BMMY medium (13.4 g/L YNB without amino acids, 20 g/L peptone, 10 g/L yeast extract, 10 g/L methanol and 100 mM potassium phosphate) according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). The MD medium plate (13.4 g/L YNB without amino acids, 20 g/L glucose, 0.4 mg/L biotin) and YPD-G418 medium plate (YPD medium with a final concentration of 1.0, 2.0 and 4.0 g/L geneticin) were used to select the Hisþ transformants and srtA multicopy transformants, respectively. As for shaking-flask SrtA expression, 12 mL/L trace elements solution PTM1 (0.2 g/L Na2MoO4$2H20, 3.0 g/L MnSO4$H20, 6.0 g/L CuSO4$5H20, 65.0 g/L FeSO4$7H20, 0.5 g/L CoCl2, 20.0 g/L ZnCl2, 0.08 g/L NaI, 0.02 g/L H3BO3, 0.2 g/L biotin and 5.0 mL/L H2SO4) in pure methanol was supplemented into BMMY medium every 24 h. 2.2. Expression of SrtA in P. pastoris The genome of S. aureus (ATCC 35556) was extracted by Genomic Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction and applied as the template for amplifying D59-srtA (forward primer: CCG GAA TTC CAA GCT AAA CCT CAA ATT CCG; reverse primer: ATA AGA ATG CGG CCG CTT AGT GGT GGT GAT GAT GAT GTT TGA CTT CTG TAG CTA CAA AGAT; restrict enzyme sites are underlined). The D59-srtA fragment were digested and inserted into the EcoR I/Not I site of pPIC9k. The plasmid (pPIC9k-D59-srtA) was transferred into E. coli JM109 cell and confirmed by DNA sequencing. As for the plasmid (pPIC9kGAPD59-srtA), the GAP promoter was amplified from genome of
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P. pastoris GS115 (forward primer: CCG GAG CTC TTT TTG TAG AAA TGT CTT GGT; reverse primer: CGC GGA TCC ATA GTT GTT CAA TTG ATT GAA ATA GG; restrict enzyme sites are underlined) and replaced the original AOX1 promoter at Sac I/BamH I site of pPIC9kD59-srtA. The correct pPIC9k-D59-srtA and pPIC9kGAP-D59-srtA plasmid were linearized at Sal I site and transformed into P. pastoris GS115, SMD1168 or KM71 cells, respectively. 2.3. Determination of srtA copy number by quantitative real-time PCR (qPCR) Genomic DNA from selected P. pastoris colonies were isolated by Genomic Extraction Kit (Qiagen, Valencia, CA) and quantified by Nanodrop ND-2000 spectrophotometer (Thermo Scientific). To detect srtA copy number, qPCR was performed using SYBR Premix Ex Taq™ Kit (Takara) and the primer sets were designed by Beacon Designer 7.0 software (srtA forward primer: CCT CAA ATT CCG AAA GAT AAA; srtA reverse primer: GTC AAT GAA AGT GTG TCC TG; ACT1 forward primer: CTC CAA TGA ACC CAA AGT CCA AC; ACT1 reverse primer: GAC AAA ACG GCC TGA ATA GAA AC). The qPCR reactions were conducted using a LightCycler 480 II Real-time PCR instrument (Roche Applied Science, Mannheim, Germany) and were carried out in final volumes of 20 mL with 10 mL of SYBR Green I Master, 0.8 mL forward and reverse primers, 1 mL diluted genomic DNA and 7.4 mL ddH2O. The program for qPCR experiment was: preincubation at 95 C for 30 s; 40 cycles of amplification step 95 C for 5 s and 60 C for 20 s; a cooling step at 50 C for 30 s; a melting curve analysis with a temperature gradient of 0.3 C/s from 50 to 95 C. The copy number of srtA gene in each strain was estimated by previous 2DDCT method [21] normalized to the endogenous reference ACT1 gene (encoding actin). All experiments were performed in biological triplicates with an independent measurement of each sample and the mean values were used for further calculations. Analysis of variance (ANOVA) was used to evaluate the difference of srtA copy number in different colonies. Data were analyzed using software SPSS 18.0 by Duncan's multiple range test. 2.4. Purification of SrtA The SrtA fermentation supernatant and Ni-NTA agarose (Qiagen) were mixed and loaded into a gravity flow column. The mixture was kept at 4 C for 4 h and then washed with a linear gradient of imidazole (10e40 mM) to eliminate contaminating proteins. The C-terminal His6-tagged SrtA was eluted from the column by 500 mM imidazole and desalted by an Amicon Ultra 3 K device (Millipore). The concentration of purified SrtA were determined by the Bradford method [22]. 2.5. SDS-page The expression of SrtA in various recombinant strains were checked by NuPAGE™ 12% Bis-Tris precast protein gels with prestained protein standard (Thermo Scientific). The bands were visualized with coomassie brilliant blue R250. 2.6. 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 mL volume of 50 mM Tris-HCl buffer (150 mM NaCl, 10 mM CaCl2, 0.5 mg substrate, pH 7.8) and 10 mL fermentation supernatant. The reactions were carried on at 37 C for 1 h by use of a Synergy H4 hybrid microplate reader (BioTek) and the fluorescence intensity (FI) was detected with 350 nm for excitation and 495 nm for recordings. One unit of SrtA activity was
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Fig. 1. The construction and selection scheme of recombinant P. pastoris strains for SrtA expression.
defined as the amount of enzyme (mg) that was able to increase of one FI per minute in the 200 mL reaction mixture. All experiments were performed in biological triplicates with an independent measurement of each sample and the mean values were used for further calculations. 2.7. Optimization of fermentation conditions To enhance extracellular expression of SrtA, the effects of induction time (24 h, 48 h, 72 h, 96 h and 120 h), induction temperature (20 C, 24 C, 28 C and 32 C), initial pH of BMMY medium (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0) and different concentrations of methanol (0.25%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5%) were investigated by single factor optimization at shaking-flask level, separately. The optimal values for each parameter were determined by the detection of extracellular SrtA activity directly. First, the induction temperature and time were comprehensively considered by the combination of each other at initial pH 7.0 (1.0% supplementary methanol). Then, applying the optimal induction temperature and time, the extracellular SrtA activities were measured at different initial pH and different concentrations of supplementary methanol, respectively. Based on these data, the best conditions for SrtA production in P. pastoris were fixed.
was performed in 200 mL volume of 50 mM Tris-HCl buffer (including 150 mM NaCl, 5 mM CaCl2, pH 7.8), 1.68 107 Gly3polystyrene beads, 10 mM purified extracellular expressed SrtA and 20 mM EGFP-LPETG. The reactions were carried out at 37 C for 1 h under vigorous shaking (250 rpm). The Gly3-polystyrene beads in the similar reaction system without SrtA were used as control. After enzymatic reaction finished, the harvested microbeads were washed three times and then resuspended in a 200 mL Tris-HCl buffer to measure the fluorescence with 488 nm for excitation and 533 nm for recordings by microplate reader. In addition, in order to calculate the efficiency of ligation between EGFP-LPETG and Gly3-polystyrene beads, the linear relation between the concentration of EGFP-LPETG and fluorescence intensity (FI) was determined. Based on the FI of different concentration of EGFP-LPETG, it was observed that their linear relation was between 5 and 60 nM (FI ¼ 185.3x-24.9, R2 ¼ 0.99; x: the concentration of EGFP-LPETG). Therefore, the average ligation efficiency of EGFP-LPETG onto Gly3-polystyrene beads mediated by SrtA was calculated from the difference of the applied and recovered concentrations of EGFP-LPETG, which was detected through the examination of their diluted FI within the scope of linear relation. 3. Results
2.8. The immobilization of EGFP on polystyrene beads catalyzed by purified extracellular expressed SrtA The Gly3-polystyrene beads and EGFP-LPETG (LPETG labeled at C-terminal) were prepared by previous method [23]. The reaction
3.1. Construct and screen the recombinant P. pastoris strain for SrtA expression In this study, the truncated D59-srtA gene was successfully
Fig. 2. SDS-Page analysis of SrtA in various supernatants. Line 1e3: GS115 strains with AOX1 promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 4e6: GS115 strains with GAP promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 7e9: KM71 strains with AOX1 promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 10e12: SMD1168 strains with AOX1 promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 13e15: SMD1168 strains with GAP promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 16e18: KM71 strains with GAP promoter from 1 g/L, 2 g/L and 3 g/L G418 plates. The black arrows point out the extracellular SrtA secreted by various P. pastoris strains.
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Fig. 3. SDS-Page detection of purified SrtA in various supernatants. Line 1: SMDAOXe3 (56.3 mg/L); Line 2: SMDGAP-3 (32.7 mg/L); Line 3: GSGAP-3 (7.8 mg/L). The black arrow points out the purified extracellular SrtA secreted by various P. pastoris strains.
inserted into the plasmid pPIC9k (with AOX1 promoter) and pPIC9kGAP (with GAP promoter). Then, the constructed plasmids were transformed into the P. pastoris hosts GS115, SMD1168 and KM71, respectively, and the positive clones were selected in plates with different concentration of geneticin (1.0, 2.0 and 3.0 g/L) (Fig. 1). The fermentation supernatants of different recombinant strains were examined by SDS-page (Fig. 2). The results showed that three strains could secrete mature SrtA (based on the calculated molecular weight 17.6 KD) into culture: (1) SMD1168 strain under control of AOX1 promoter from 3.0 g/L geneticin plate (SMDAOX-3); (2) SMD1168 strain under control of GAP promoter from 3.0 g/L geneticin plate (SMDGAP-3); (3) GS115 strain under control of GAP promoter from 3.0 g/L geneticin plate (GSGAP-3). In addition, based on the Ni-NTA purification, the extracellular SrtA concentration was 56.3, 32.7 and 7.8 mg/L for these three strains (Fig. 3). 3.2. Confirm the best strain for SrtA expression by enzymatic assay and gene copy number determination Besides the expression level of SrtA, its enzymatic activity was more crucial for the further application in ligation reactions. The
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€rster resonance energy transfer) substrate of SrtA specific FRET (Fo was used to confirm the extracellular activity for all recombinant P. pastoris strains directly. As shown in Table 1, the activity of SrtA could be detected in five fermentation supernatants, which was highly consistent with its expression level. Among these four strains, the extracellular SrtA activity of SMDAOX-3 (83.1 U/mL) and SMDGAP-3 (42.6 U/mL) were much higher than others. Furthermore, as SMDAOX-3 was selected from the 3.0 g/L geneticin plate, it is necessary to examine the real srtA copy number in its chromosome. The SMDAOX-3 strain was recoated on a plate with 3.0 g/L geneticin and ten colonies were randomly chosen to determine their srtA copy number by qRT-PCR. According to the method for relative quantification, the results showed that the difference of srtA copy number between ten colonies of SMDAOX-3 strain was very small (p > 0.05). The average of srtA copies in ten colonies was 5.7 (Figure S1). Based on the best enzymatic activity and the integrated srtA copies, SMDAOX-3 was the best strain for SrtA expression in P. pastoris. 3.3. Optimization of the conditions for SrtA production in P. pastoris SMDAOX-3 could secrete more SrtA than other P. pastoris strains, but the yield of extracellular SrtA (56.3 mg/L) was not as high as the yields reported in previous researches [13]. Therefore, fermentation process optimization was investigated to enhance its yield. Four important conditions, including induction temperature, time, pH and methanol concentration, were optimized at shaking-flask level by one-factor-at-a-time approach. At first, Different induction temperatures (20 C, 24 C, 28 C and 32 C) and induction time (24 h, 48 h, 72 h, 96 h and 120 h) were combined to attempt with constant induction pH (7.0) and methanol concentration (1.0%). The results showed that the best enzymatic activity reached when temperature maintained in 28 C (Fig. 4A). Although the enzymatic activity was a little higher in 96 h (109.3 U/mL) than 72 h (105.4 U/ mL), the productivity in 96 h (1.1 U/mL/h) was lower than 72 h (1.5 U/mL/h). Therefore, considering the productivity, production cost and the balance between longer duration and satisfactory amount of activity, 72 h was chosen as the optimal induction time. As for the initial pH (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0), the preferable initial pH for SrtA expression was pH 6.0 (Fig. 4B). Furthermore, the effect of methanol concentration (0.25%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5%) on SrtA production was investigated and the results showed
Table 1 The extracellular activity and concentration of SrtA for all recombinant P. pastoris strains. Host
Promoter
G418 resistance (g/L)
Extracellular SrtA Activity (U/mL)
Concentration (mg/L)
GS115
AOX1
1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0
-a e 1.1 e e 9.6 e e 83.1 e e 42.6 e 1.6 e e e e
e e 2.1 e e 7.8 e 1.3 56.3 e 1.1 32.7 e 4.3 e 1.5 e e
GAP
SMD1168
AOX1
GAP
KM71
AOX1
GAP
a
“-” means no extracellular activity or concentration of SrtA was detected.
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1.5% (v/v) methanol was the most suitable inducing concentration (Fig. 4C). Finally, by combining the best fermentation parameters, the expression level of extracellular SrtA reached 97.8 mg/L (Table 2) and the biomass (OD600) reach 5.2 at shaking-flask level. This expression level was 27.3% increase compared with the previous highest intracellular expression level (76.9 mg/L) [13]. The optimized results showed that the best conditions for SrtA production in P. pastoris were as follow: the cells were cultured in BMMY medium (pH 6.0) supplemented with 1.5% methanol and grown 72 h at 28 C. 3.4. The transpeptidation activity of extracellular expressed SrtA confirmed by the reaction between Gly3-polystyrene and EGFPLPETG Applying purified extracellular expressed SrtA (10 mM), the ligation reaction between EGFP-LPETG and Gly3-polystyrene beads was finished within 1 h. After catalyzed by SrtA, the fluorescence intensity of EGFP-LPET-Gly3-polystyrene was significantly increased compared with the control (Gly3-polystyrene beads and EGFP-LPETG without SrtA) (Fig. 5A and B). In addition, according to the good linear relation between the concentration of EGFP-LPETG and fluorescence intensity (5e60 nM) (Fig. 5C), the ligation efficiency was 24.3%. 4. Discussion
Fig. 4. The effect of induction time, induction temperature, initial pH and methanol concentration on the production of extracellular SrtA. (A) induction time and induction temperature; (B) initial pH; (C) methanol concentration. Each value represents the mean of three independent measurements and the deviation from the mean is<5%.
In this study, three P. pastoris strains (GS115, SMD1168 and KM71) were chosen to obtain the best strategy for SrtA extracellular expression. The choice of proper strain is important for the expression of heterologous proteins or enzymes. The strain GS115 with clear genetic background can utilize methanol by a functional copy of the alcohol oxidase 1 gene (AOX1). The strain SMD1168 also can utilize methanol, but it lacks the vacuole peptidase A (Pep4), which efficiently reduce the proteolysis in the host. As for the strain KM71, it is a methanol-utilization slow (MutS) phenotype and is not sensitive to the residual methanol in the media [14]. According to the results for SrtA expression, P. pastoris SMD1168 was the most suitable host based on the expression level and extracellular enzymatic activity. It means that the existence of protease may have adverse effect on the stability and secretion of SrtA [24]. In addition, since many proteins have been successfully expressed by either AOX1 or GAP promoter in P. pastoris [25], both promoters (SMDAOX-3 and SMDGAP-3) were tested for SrtA extracellular expression. The results of activity assays demonstrated that although both two promoters can mediate SrtA expression, AOX1 promoter was more efficient than GAP promoter. Moreover, as all SrtA-expressed strains were obtained from the plates with high concentration of geneticin, which was in accord with the introduction of vector pPIC9k (one copy of integrated foreign gene bring approximate 0.5 g/L geneticin resistance to the host) [26]. It suggests that the multicopy of srtA was essential for its extracellular expression because the increase in copy number of integrated gene could result in a proportional elevation in the transcription and expression level of heterologous protein [27]. Based on the best recombinant strain (SMDAOX-3), the suitable fermentation conditions for SrtA extracellular expression were
Table 2 The purification summary table for extracellular SrtA from 1 L fermentation broth. Steps
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purity (fold)
Culture supernatant Ni affinity chromatography Desalination Freeze-drying
1205.3 128.6 105.3 97.8
199510 146320 139790 131900
165.5 1137.8 1327.5 1348.7
100 73.3 70.1 66.1
1 6.9 8.0 8.1
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5. Conclusion In this study, the efficient extracellular expression of SrtA was achieved in the P. pastoris SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter. Based on the optimal fermentation conditions (induction 72 h at 28 C, initial pH 6.0, supplemented with 1.5% methanol), the highest expression level and activity of extracellular SrtA could reach 97.8 mg/L and 131.9 U/ mL, respectively. In addition, the ligation reaction between EGFP and Gly3-polystyrene beads could be catalyzed by extracellular purified SrtA (efficiency: 24.3%). Author agreement All authors have read and approved to submit it to your journal. There is no conflict of interest of any author in relation to the submission. This paper has not been submitted elsewhere for consideration of publication. Competing financial interests The authors declare no competing financial interests. Author contributions Zhimeng Wu designed experiments; Haofei Hong performed some shaking-flask culture tests; Xinrui Zhao carried out experiments, analyzed experimental results, wrote the main manuscript text and prepared all tables and figures in this manuscript. All authors reviewed the manuscript. Fig. 5. The ligation reaction between EGFP-LPETG and Gly3-polystyrene beads catalyzed by extracellular SrtA. (A). The fluorescence image and intensity of EGFPLPET-Gly3-polystyrene beads (catalyzed by SrtA); (B) The fluorescence image and intensity of Gly3-polystyrene beads mixed with EGFP-LPETG without SrtA (control); (C) The linear relation between the concentration of EGFP-LPETG and fluorescence intensity (5e60 nmol).
fixed at shaking-flask level. As induction temperature and time are critical parameters for any P. pastoris fermentation, control of them is important for maximizing expression level [28]. Although P. pastoris can grow in the range of 20e30 C, suitable temperature can increase carbon flux toward protein expression and preventing byproduct formation. The optimal results obtained in this research indicated that the moderate induction temperature is important for SrtA production to prevent fast growth rates that lead to cell lysis [29] and the extending induction time is not necessary as there was no significant increase in enzymatic activity after 72 h induction fermentation. As for optimal pH, although P. pastoris is able to grow and express foreign proteins in a wide range of pH (3.0e8.0) [30], the preferable induction pH for SrtA expression was pH 6.0. Applying the best strain and conditions, the expression level of extracellular SrtA could reach 97.8 mg/L, which was 27.3% increase compared with the previous highest intracellular expression level (76.9 mg/L) [13]. Not only was the expression level of SrtA significantly improved, but also the purification step for extracellular SrtA was simplified. Therefore, the better strategy and conditions for SrtA fermentation have a great potential in industrial production in the future. To confirm extracellular SrtA transpeptidation activity, the immobilization of EGFP-LPETG to glycine modified polystyrene beads was performed. Ligation efficiency (24.3%) can be easily measured by detecting the fluorescence intensity of EGFP. The result indicated that extracellular SrtA could immobilize EGFPLPETG onto the surface of Gly3-polystyrene directly.
Acknowledgment This work was supported by the National Natural Science Foundation of China (21472070), the Project for Jiangsu Scientific and Technological Innovation Team, Fund for Jiangsu Distinguished Professorship Program, Jiangsu Postdoctoral Science Foundation (1402070C), and the State Key Laboratory of Natural and Biomimetic Drugs (K20140216). Project 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. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pep.2017.03.010. References [1] S.K. Mazmanian, G. Liu, H. Ton-That, O. Schneewind, Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall, Science 285 (1999) 760e763. [2] H. Ton-That, S.K. Mazmanian, K.F. Faull, O. Schneewind, Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates, J. Biol. Chem. 275 (2000) 9876e9881. [3] D.A. Levary, R. Parthasarathy, E.T. Boder, M.E. Ackerman, Protein-protein fusion catalyzed by sortase A, PLoS One 6 (2011) e18342. [4] Q. Sun, Q. Chen, D. Blackstock, W. Chen, Post-translational modification of bionanoparticles as a modular platform for biosensor assembly, ACS Nano 9 (2015) 8554e8561. [5] Z. Wu, X. Guo, Z. Guo, Sortase A-catalyzed peptide cyclization for the synthesis of macrocyclic peptides and glycopeptides, Chem. Commun. 47 (2011) 9218e9220. [6] X. Guo, Q. Wang, B.M. Swarts, Z. Guo, Sortase-catalyzed peptideglycosylphosphatidylinositol analogue ligation, J. Am. Chem. Soc. 131 (2009) 9878e9879.
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Efficient extracellular expression of transpeptidase sortase A in Pichia pastoris Xinrui Zhao a, Haofei Hong a, Zhimeng Wu a, b, * a The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road, Wuxi 1800, China b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2016 Received in revised form 28 February 2017 Accepted 13 March 2017 Available online 15 March 2017
In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly3polystyrene beads. © 2017 Elsevier Inc. All rights reserved.
Keywords: Sortase A Extracellular expression Immobilization Optimization Pichia pastoris
1. Introduction Sortase A (SrtA, EC: 3.4.22.70) is a transpeptidase found in Staphylococcus aureus [1]. It anchors surface proteins with a specific C-terminal sorting signal (LPXTG, X stand for any amino acid except cysteine) to cell wall by cleaving the amide bond between threonine and glycine and transferring the thioester intermediate to the amino group of pentaglycine cross-bridges [2]. Due to its sitespecific transpeptidation reaction, SrtA has been widely used as a ligation tool in the fusion of protein domains with their preserved functionality [3], post-translational modification of bionanoparticles [4], peptides and protein cyclization [5], preparation of complex glycoconjugates [6] and live cell labelling [7]. Especially, SrtAmediated ligation was applied to synthesize antibody-drug conjugates as highly promising biotherapeutics [8]. Furthermore, efforts to evolve SrtA by site-mutations have generated variants of SrtA with improved kinetics, which were applied in antibody and protein conjugation reactions [9,10]. Thus, SrtA has great potential applications in biotechnology industry.
* Corresponding author. The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, China. E-mail address: [email protected] (Z. Wu). http://dx.doi.org/10.1016/j.pep.2017.03.010 1046-5928/© 2017 Elsevier Inc. All rights reserved.
So far, expression of SrtA has long been limited in intracellular expression in Escherichia coli by using various commercial plasmids, including pET23b (Novagen) [6], pQE30 (Qiagen) [11] and pBAD (Invitrogen) [12] etc.. Although these strategies could produce SrtA for the laboratory use, the expression level of SrtA maintained at low level with yields ranging from several milligrams to maximum 76.9 mg/L [13]. Disadvantages of these intracellular expression strategies, including cumbrous cell disruption and timeconsuming downstream purification process, obstructed its future industrial application. In addition, compared with the extensive applications of SrtA, few endeavors were made to improve or optimize the expression level of this important enzyme. Therefore, new strategy for the efficient extracellular expression of SrtA is highly demanded. In the last two decades, the methylotrophic Pichia pastoris has proven to be an excellent host for the production of a variety of heterologous proteins in both academia and industry. This expression system featured with ease genetic manipulation, high cell densities using inexpensive medium and post-translational modification. In addition, various P. pastoris hosts, such as GS115 (Mutþ phenotype), SMD1168 (Mutþ phenotype, defective in the vacuole peptidase A) and KM71 (Muts phenotype) etc. [14], and promoters (including the methanol-induced AOX1 promoter and constitutive GAP promoter etc.) [15] were available for screening the specific expression requirement. In particular, with the
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successful a-factor signal peptide from Saccharomyces cerevisiae [16], many large-scale extracellular production of recombinant proteins was achieved at high levels in P. pastoris [17]. This a-factor signal peptide can efficiently mediate the secretion of heterologous proteins through endoplasmic reticulum, Golgi apparatus and plasma membrane [18]. Furthermore, target protein was harvested from the culture medium directly without cell-disruption or refolding operation, which greatly simplify the downstream purification steps [19]. Therefore, the P. pastoris system has a potential for the efficient extracellular expression of SrtA. In this study, extracellular expression of a truncated version of SrtA (D59-SrtA with good solubility and the same transpeptidation activity with full-length SrtA) [20] in P. pastoris was investigated. By adopting various strategies (different hosts, promoters and gene copy numbers) for protein expression in P. pastoris, a series of recombinant engineered strains were constructed and the extracellular expression of SrtA was firstly achieved. Based on SDS-page analysis and activity assay, a suitable strain to secret expression of SrtA was obtained. Then, the expression level of SrtA was further enhanced by the optimization of fermentation conditions. Finally, the transpeptidation activity of secreted SrtA was confirmed by immobilization of enhanced green fluorescent protein (EGFP) onto glycine-modified polystyrene beads. 2. Materials and methods 2.1. Strain, plasmid and media The P. pastoris host strain GS115 (his4, Mutþ, His-, aox1þ, aox2þ), SMD1168 (his4, pep4, Mutþ, His-, aox1þ, aox2þ) and KM71 (his4, MutS, His-, aox1-, aox2þ) and plasmid pPIC9k were purchased from Invitrogen (Carlsbad, USA). The E. coli JM109 (Novagen, Madison, WI) was used as the host for cloning and DNA sequencing. E. coli strain was cultured in LB medium (Tryptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L) with 100 mg/mL ampicillin. P. pastoris strains were grown in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose), BMGY medium (13.4 g/L YNB without amino acids, 20 g/L peptone, 10 g/L yeast extract, 10 g/L glycerol and 100 mM potassium phosphate) or BMMY medium (13.4 g/L YNB without amino acids, 20 g/L peptone, 10 g/L yeast extract, 10 g/L methanol and 100 mM potassium phosphate) according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). The MD medium plate (13.4 g/L YNB without amino acids, 20 g/L glucose, 0.4 mg/L biotin) and YPD-G418 medium plate (YPD medium with a final concentration of 1.0, 2.0 and 4.0 g/L geneticin) were used to select the Hisþ transformants and srtA multicopy transformants, respectively. As for shaking-flask SrtA expression, 12 mL/L trace elements solution PTM1 (0.2 g/L Na2MoO4$2H20, 3.0 g/L MnSO4$H20, 6.0 g/L CuSO4$5H20, 65.0 g/L FeSO4$7H20, 0.5 g/L CoCl2, 20.0 g/L ZnCl2, 0.08 g/L NaI, 0.02 g/L H3BO3, 0.2 g/L biotin and 5.0 mL/L H2SO4) in pure methanol was supplemented into BMMY medium every 24 h. 2.2. Expression of SrtA in P. pastoris The genome of S. aureus (ATCC 35556) was extracted by Genomic Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction and applied as the template for amplifying D59-srtA (forward primer: CCG GAA TTC CAA GCT AAA CCT CAA ATT CCG; reverse primer: ATA AGA ATG CGG CCG CTT AGT GGT GGT GAT GAT GAT GTT TGA CTT CTG TAG CTA CAA AGAT; restrict enzyme sites are underlined). The D59-srtA fragment were digested and inserted into the EcoR I/Not I site of pPIC9k. The plasmid (pPIC9k-D59-srtA) was transferred into E. coli JM109 cell and confirmed by DNA sequencing. As for the plasmid (pPIC9kGAPD59-srtA), the GAP promoter was amplified from genome of
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P. pastoris GS115 (forward primer: CCG GAG CTC TTT TTG TAG AAA TGT CTT GGT; reverse primer: CGC GGA TCC ATA GTT GTT CAA TTG ATT GAA ATA GG; restrict enzyme sites are underlined) and replaced the original AOX1 promoter at Sac I/BamH I site of pPIC9kD59-srtA. The correct pPIC9k-D59-srtA and pPIC9kGAP-D59-srtA plasmid were linearized at Sal I site and transformed into P. pastoris GS115, SMD1168 or KM71 cells, respectively. 2.3. Determination of srtA copy number by quantitative real-time PCR (qPCR) Genomic DNA from selected P. pastoris colonies were isolated by Genomic Extraction Kit (Qiagen, Valencia, CA) and quantified by Nanodrop ND-2000 spectrophotometer (Thermo Scientific). To detect srtA copy number, qPCR was performed using SYBR Premix Ex Taq™ Kit (Takara) and the primer sets were designed by Beacon Designer 7.0 software (srtA forward primer: CCT CAA ATT CCG AAA GAT AAA; srtA reverse primer: GTC AAT GAA AGT GTG TCC TG; ACT1 forward primer: CTC CAA TGA ACC CAA AGT CCA AC; ACT1 reverse primer: GAC AAA ACG GCC TGA ATA GAA AC). The qPCR reactions were conducted using a LightCycler 480 II Real-time PCR instrument (Roche Applied Science, Mannheim, Germany) and were carried out in final volumes of 20 mL with 10 mL of SYBR Green I Master, 0.8 mL forward and reverse primers, 1 mL diluted genomic DNA and 7.4 mL ddH2O. The program for qPCR experiment was: preincubation at 95 C for 30 s; 40 cycles of amplification step 95 C for 5 s and 60 C for 20 s; a cooling step at 50 C for 30 s; a melting curve analysis with a temperature gradient of 0.3 C/s from 50 to 95 C. The copy number of srtA gene in each strain was estimated by previous 2DDCT method [21] normalized to the endogenous reference ACT1 gene (encoding actin). All experiments were performed in biological triplicates with an independent measurement of each sample and the mean values were used for further calculations. Analysis of variance (ANOVA) was used to evaluate the difference of srtA copy number in different colonies. Data were analyzed using software SPSS 18.0 by Duncan's multiple range test. 2.4. Purification of SrtA The SrtA fermentation supernatant and Ni-NTA agarose (Qiagen) were mixed and loaded into a gravity flow column. The mixture was kept at 4 C for 4 h and then washed with a linear gradient of imidazole (10e40 mM) to eliminate contaminating proteins. The C-terminal His6-tagged SrtA was eluted from the column by 500 mM imidazole and desalted by an Amicon Ultra 3 K device (Millipore). The concentration of purified SrtA were determined by the Bradford method [22]. 2.5. SDS-page The expression of SrtA in various recombinant strains were checked by NuPAGE™ 12% Bis-Tris precast protein gels with prestained protein standard (Thermo Scientific). The bands were visualized with coomassie brilliant blue R250. 2.6. 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 mL volume of 50 mM Tris-HCl buffer (150 mM NaCl, 10 mM CaCl2, 0.5 mg substrate, pH 7.8) and 10 mL fermentation supernatant. The reactions were carried on at 37 C for 1 h by use of a Synergy H4 hybrid microplate reader (BioTek) and the fluorescence intensity (FI) was detected with 350 nm for excitation and 495 nm for recordings. One unit of SrtA activity was
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Fig. 1. The construction and selection scheme of recombinant P. pastoris strains for SrtA expression.
defined as the amount of enzyme (mg) that was able to increase of one FI per minute in the 200 mL reaction mixture. All experiments were performed in biological triplicates with an independent measurement of each sample and the mean values were used for further calculations. 2.7. Optimization of fermentation conditions To enhance extracellular expression of SrtA, the effects of induction time (24 h, 48 h, 72 h, 96 h and 120 h), induction temperature (20 C, 24 C, 28 C and 32 C), initial pH of BMMY medium (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0) and different concentrations of methanol (0.25%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5%) were investigated by single factor optimization at shaking-flask level, separately. The optimal values for each parameter were determined by the detection of extracellular SrtA activity directly. First, the induction temperature and time were comprehensively considered by the combination of each other at initial pH 7.0 (1.0% supplementary methanol). Then, applying the optimal induction temperature and time, the extracellular SrtA activities were measured at different initial pH and different concentrations of supplementary methanol, respectively. Based on these data, the best conditions for SrtA production in P. pastoris were fixed.
was performed in 200 mL volume of 50 mM Tris-HCl buffer (including 150 mM NaCl, 5 mM CaCl2, pH 7.8), 1.68 107 Gly3polystyrene beads, 10 mM purified extracellular expressed SrtA and 20 mM EGFP-LPETG. The reactions were carried out at 37 C for 1 h under vigorous shaking (250 rpm). The Gly3-polystyrene beads in the similar reaction system without SrtA were used as control. After enzymatic reaction finished, the harvested microbeads were washed three times and then resuspended in a 200 mL Tris-HCl buffer to measure the fluorescence with 488 nm for excitation and 533 nm for recordings by microplate reader. In addition, in order to calculate the efficiency of ligation between EGFP-LPETG and Gly3-polystyrene beads, the linear relation between the concentration of EGFP-LPETG and fluorescence intensity (FI) was determined. Based on the FI of different concentration of EGFP-LPETG, it was observed that their linear relation was between 5 and 60 nM (FI ¼ 185.3x-24.9, R2 ¼ 0.99; x: the concentration of EGFP-LPETG). Therefore, the average ligation efficiency of EGFP-LPETG onto Gly3-polystyrene beads mediated by SrtA was calculated from the difference of the applied and recovered concentrations of EGFP-LPETG, which was detected through the examination of their diluted FI within the scope of linear relation. 3. Results
2.8. The immobilization of EGFP on polystyrene beads catalyzed by purified extracellular expressed SrtA The Gly3-polystyrene beads and EGFP-LPETG (LPETG labeled at C-terminal) were prepared by previous method [23]. The reaction
3.1. Construct and screen the recombinant P. pastoris strain for SrtA expression In this study, the truncated D59-srtA gene was successfully
Fig. 2. SDS-Page analysis of SrtA in various supernatants. Line 1e3: GS115 strains with AOX1 promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 4e6: GS115 strains with GAP promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 7e9: KM71 strains with AOX1 promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 10e12: SMD1168 strains with AOX1 promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 13e15: SMD1168 strains with GAP promoter from 1 g/L, 2 g/L and 3 g/L G418 plates; Line 16e18: KM71 strains with GAP promoter from 1 g/L, 2 g/L and 3 g/L G418 plates. The black arrows point out the extracellular SrtA secreted by various P. pastoris strains.
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Fig. 3. SDS-Page detection of purified SrtA in various supernatants. Line 1: SMDAOXe3 (56.3 mg/L); Line 2: SMDGAP-3 (32.7 mg/L); Line 3: GSGAP-3 (7.8 mg/L). The black arrow points out the purified extracellular SrtA secreted by various P. pastoris strains.
inserted into the plasmid pPIC9k (with AOX1 promoter) and pPIC9kGAP (with GAP promoter). Then, the constructed plasmids were transformed into the P. pastoris hosts GS115, SMD1168 and KM71, respectively, and the positive clones were selected in plates with different concentration of geneticin (1.0, 2.0 and 3.0 g/L) (Fig. 1). The fermentation supernatants of different recombinant strains were examined by SDS-page (Fig. 2). The results showed that three strains could secrete mature SrtA (based on the calculated molecular weight 17.6 KD) into culture: (1) SMD1168 strain under control of AOX1 promoter from 3.0 g/L geneticin plate (SMDAOX-3); (2) SMD1168 strain under control of GAP promoter from 3.0 g/L geneticin plate (SMDGAP-3); (3) GS115 strain under control of GAP promoter from 3.0 g/L geneticin plate (GSGAP-3). In addition, based on the Ni-NTA purification, the extracellular SrtA concentration was 56.3, 32.7 and 7.8 mg/L for these three strains (Fig. 3). 3.2. Confirm the best strain for SrtA expression by enzymatic assay and gene copy number determination Besides the expression level of SrtA, its enzymatic activity was more crucial for the further application in ligation reactions. The
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€rster resonance energy transfer) substrate of SrtA specific FRET (Fo was used to confirm the extracellular activity for all recombinant P. pastoris strains directly. As shown in Table 1, the activity of SrtA could be detected in five fermentation supernatants, which was highly consistent with its expression level. Among these four strains, the extracellular SrtA activity of SMDAOX-3 (83.1 U/mL) and SMDGAP-3 (42.6 U/mL) were much higher than others. Furthermore, as SMDAOX-3 was selected from the 3.0 g/L geneticin plate, it is necessary to examine the real srtA copy number in its chromosome. The SMDAOX-3 strain was recoated on a plate with 3.0 g/L geneticin and ten colonies were randomly chosen to determine their srtA copy number by qRT-PCR. According to the method for relative quantification, the results showed that the difference of srtA copy number between ten colonies of SMDAOX-3 strain was very small (p > 0.05). The average of srtA copies in ten colonies was 5.7 (Figure S1). Based on the best enzymatic activity and the integrated srtA copies, SMDAOX-3 was the best strain for SrtA expression in P. pastoris. 3.3. Optimization of the conditions for SrtA production in P. pastoris SMDAOX-3 could secrete more SrtA than other P. pastoris strains, but the yield of extracellular SrtA (56.3 mg/L) was not as high as the yields reported in previous researches [13]. Therefore, fermentation process optimization was investigated to enhance its yield. Four important conditions, including induction temperature, time, pH and methanol concentration, were optimized at shaking-flask level by one-factor-at-a-time approach. At first, Different induction temperatures (20 C, 24 C, 28 C and 32 C) and induction time (24 h, 48 h, 72 h, 96 h and 120 h) were combined to attempt with constant induction pH (7.0) and methanol concentration (1.0%). The results showed that the best enzymatic activity reached when temperature maintained in 28 C (Fig. 4A). Although the enzymatic activity was a little higher in 96 h (109.3 U/mL) than 72 h (105.4 U/ mL), the productivity in 96 h (1.1 U/mL/h) was lower than 72 h (1.5 U/mL/h). Therefore, considering the productivity, production cost and the balance between longer duration and satisfactory amount of activity, 72 h was chosen as the optimal induction time. As for the initial pH (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0), the preferable initial pH for SrtA expression was pH 6.0 (Fig. 4B). Furthermore, the effect of methanol concentration (0.25%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5%) on SrtA production was investigated and the results showed
Table 1 The extracellular activity and concentration of SrtA for all recombinant P. pastoris strains. Host
Promoter
G418 resistance (g/L)
Extracellular SrtA Activity (U/mL)
Concentration (mg/L)
GS115
AOX1
1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0
-a e 1.1 e e 9.6 e e 83.1 e e 42.6 e 1.6 e e e e
e e 2.1 e e 7.8 e 1.3 56.3 e 1.1 32.7 e 4.3 e 1.5 e e
GAP
SMD1168
AOX1
GAP
KM71
AOX1
GAP
a
“-” means no extracellular activity or concentration of SrtA was detected.
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1.5% (v/v) methanol was the most suitable inducing concentration (Fig. 4C). Finally, by combining the best fermentation parameters, the expression level of extracellular SrtA reached 97.8 mg/L (Table 2) and the biomass (OD600) reach 5.2 at shaking-flask level. This expression level was 27.3% increase compared with the previous highest intracellular expression level (76.9 mg/L) [13]. The optimized results showed that the best conditions for SrtA production in P. pastoris were as follow: the cells were cultured in BMMY medium (pH 6.0) supplemented with 1.5% methanol and grown 72 h at 28 C. 3.4. The transpeptidation activity of extracellular expressed SrtA confirmed by the reaction between Gly3-polystyrene and EGFPLPETG Applying purified extracellular expressed SrtA (10 mM), the ligation reaction between EGFP-LPETG and Gly3-polystyrene beads was finished within 1 h. After catalyzed by SrtA, the fluorescence intensity of EGFP-LPET-Gly3-polystyrene was significantly increased compared with the control (Gly3-polystyrene beads and EGFP-LPETG without SrtA) (Fig. 5A and B). In addition, according to the good linear relation between the concentration of EGFP-LPETG and fluorescence intensity (5e60 nM) (Fig. 5C), the ligation efficiency was 24.3%. 4. Discussion
Fig. 4. The effect of induction time, induction temperature, initial pH and methanol concentration on the production of extracellular SrtA. (A) induction time and induction temperature; (B) initial pH; (C) methanol concentration. Each value represents the mean of three independent measurements and the deviation from the mean is<5%.
In this study, three P. pastoris strains (GS115, SMD1168 and KM71) were chosen to obtain the best strategy for SrtA extracellular expression. The choice of proper strain is important for the expression of heterologous proteins or enzymes. The strain GS115 with clear genetic background can utilize methanol by a functional copy of the alcohol oxidase 1 gene (AOX1). The strain SMD1168 also can utilize methanol, but it lacks the vacuole peptidase A (Pep4), which efficiently reduce the proteolysis in the host. As for the strain KM71, it is a methanol-utilization slow (MutS) phenotype and is not sensitive to the residual methanol in the media [14]. According to the results for SrtA expression, P. pastoris SMD1168 was the most suitable host based on the expression level and extracellular enzymatic activity. It means that the existence of protease may have adverse effect on the stability and secretion of SrtA [24]. In addition, since many proteins have been successfully expressed by either AOX1 or GAP promoter in P. pastoris [25], both promoters (SMDAOX-3 and SMDGAP-3) were tested for SrtA extracellular expression. The results of activity assays demonstrated that although both two promoters can mediate SrtA expression, AOX1 promoter was more efficient than GAP promoter. Moreover, as all SrtA-expressed strains were obtained from the plates with high concentration of geneticin, which was in accord with the introduction of vector pPIC9k (one copy of integrated foreign gene bring approximate 0.5 g/L geneticin resistance to the host) [26]. It suggests that the multicopy of srtA was essential for its extracellular expression because the increase in copy number of integrated gene could result in a proportional elevation in the transcription and expression level of heterologous protein [27]. Based on the best recombinant strain (SMDAOX-3), the suitable fermentation conditions for SrtA extracellular expression were
Table 2 The purification summary table for extracellular SrtA from 1 L fermentation broth. Steps
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purity (fold)
Culture supernatant Ni affinity chromatography Desalination Freeze-drying
1205.3 128.6 105.3 97.8
199510 146320 139790 131900
165.5 1137.8 1327.5 1348.7
100 73.3 70.1 66.1
1 6.9 8.0 8.1
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5. Conclusion In this study, the efficient extracellular expression of SrtA was achieved in the P. pastoris SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter. Based on the optimal fermentation conditions (induction 72 h at 28 C, initial pH 6.0, supplemented with 1.5% methanol), the highest expression level and activity of extracellular SrtA could reach 97.8 mg/L and 131.9 U/ mL, respectively. In addition, the ligation reaction between EGFP and Gly3-polystyrene beads could be catalyzed by extracellular purified SrtA (efficiency: 24.3%). Author agreement All authors have read and approved to submit it to your journal. There is no conflict of interest of any author in relation to the submission. This paper has not been submitted elsewhere for consideration of publication. Competing financial interests The authors declare no competing financial interests. Author contributions Zhimeng Wu designed experiments; Haofei Hong performed some shaking-flask culture tests; Xinrui Zhao carried out experiments, analyzed experimental results, wrote the main manuscript text and prepared all tables and figures in this manuscript. All authors reviewed the manuscript. Fig. 5. The ligation reaction between EGFP-LPETG and Gly3-polystyrene beads catalyzed by extracellular SrtA. (A). The fluorescence image and intensity of EGFPLPET-Gly3-polystyrene beads (catalyzed by SrtA); (B) The fluorescence image and intensity of Gly3-polystyrene beads mixed with EGFP-LPETG without SrtA (control); (C) The linear relation between the concentration of EGFP-LPETG and fluorescence intensity (5e60 nmol).
fixed at shaking-flask level. As induction temperature and time are critical parameters for any P. pastoris fermentation, control of them is important for maximizing expression level [28]. Although P. pastoris can grow in the range of 20e30 C, suitable temperature can increase carbon flux toward protein expression and preventing byproduct formation. The optimal results obtained in this research indicated that the moderate induction temperature is important for SrtA production to prevent fast growth rates that lead to cell lysis [29] and the extending induction time is not necessary as there was no significant increase in enzymatic activity after 72 h induction fermentation. As for optimal pH, although P. pastoris is able to grow and express foreign proteins in a wide range of pH (3.0e8.0) [30], the preferable induction pH for SrtA expression was pH 6.0. Applying the best strain and conditions, the expression level of extracellular SrtA could reach 97.8 mg/L, which was 27.3% increase compared with the previous highest intracellular expression level (76.9 mg/L) [13]. Not only was the expression level of SrtA significantly improved, but also the purification step for extracellular SrtA was simplified. Therefore, the better strategy and conditions for SrtA fermentation have a great potential in industrial production in the future. To confirm extracellular SrtA transpeptidation activity, the immobilization of EGFP-LPETG to glycine modified polystyrene beads was performed. Ligation efficiency (24.3%) can be easily measured by detecting the fluorescence intensity of EGFP. The result indicated that extracellular SrtA could immobilize EGFPLPETG onto the surface of Gly3-polystyrene directly.
Acknowledgment This work was supported by the National Natural Science Foundation of China (21472070), the Project for Jiangsu Scientific and Technological Innovation Team, Fund for Jiangsu Distinguished Professorship Program, Jiangsu Postdoctoral Science Foundation (1402070C), and the State Key Laboratory of Natural and Biomimetic Drugs (K20140216). Project 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. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pep.2017.03.010. References [1] S.K. Mazmanian, G. Liu, H. Ton-That, O. Schneewind, Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall, Science 285 (1999) 760e763. [2] H. Ton-That, S.K. Mazmanian, K.F. Faull, O. Schneewind, Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates, J. Biol. Chem. 275 (2000) 9876e9881. [3] D.A. Levary, R. Parthasarathy, E.T. Boder, M.E. Ackerman, Protein-protein fusion catalyzed by sortase A, PLoS One 6 (2011) e18342. [4] Q. Sun, Q. Chen, D. Blackstock, W. Chen, Post-translational modification of bionanoparticles as a modular platform for biosensor assembly, ACS Nano 9 (2015) 8554e8561. [5] Z. Wu, X. Guo, Z. Guo, Sortase A-catalyzed peptide cyclization for the synthesis of macrocyclic peptides and glycopeptides, Chem. Commun. 47 (2011) 9218e9220. [6] X. Guo, Q. Wang, B.M. Swarts, Z. Guo, Sortase-catalyzed peptideglycosylphosphatidylinositol analogue ligation, J. Am. Chem. Soc. 131 (2009) 9878e9879.
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