Active non-receptor Src protein tyrosine kinases are usually expressed with very low yield due to protein toxicity associated with its kinase activity. In this study, Src was expressed by means of Protein Tyrosine Phosphatase 1B (PTP1B) to facilitate the production of active Src by neutralizing their apparent toxicity to cells in Pichia pastoris. Several recombinant Src-producing strains were constructed, and they displayed the catalytic activity of protein tyrosine phosphatase. The plasmid with c-Src fused with a his-tag and Green Fluorescent Protein (GFP) was transformed into P. pastoris with/without PTP1B, resulting in five recombinants with different expression levels. The c-Src fusion protein was purified by Ni2+ affinity chromatography. Among these recombinant strains, the c-Src co-expressed with PTP1B exhibited higher kinase catalytic activity than those expressing the c-Src alone. Consequently, the c-Src fusion protein expressed in P. pastoris might be attractive drug discovering targets against cancer.
Keywords: c-Src; Protein Expression; Protein tyrosine kinase; Protein Tyrosine Phosphatase; Pichia pastoris
The Src family of protein tyrosine kinases (PTK) comprises a transforming protein v-Src and its cellular proto-oncogene homolog c-Src . Src has been extensively studied for its important role in normal cellular signaling and tumorigenesis . Significant progress has been made in resolution of Src structure, identification of physiological substrates, characterization of Src signaling pathways, and elucidation of its roles in tumorigenesis and osteoporosis [3-5]. Considerable evidence has now demonstrated that Src is an important target for drug discovery against cancer and osteoporosis . However, the questions regarding the detail molecular mechanisms of Src catalysis and regulation still remain unknown. In order to answer the questions, the biochemical and structural studies have to be combined during which the recombinant protein preparation and the site-specific mutagenesis are often demanded. Although the recombinant Src has been successfully expressed in several eukaryotic systems [7,8], such expression systems are inefficient and faultily suited for large-scale mutagenesis studies because of low Src yield and/or just domain expression.
Protein tyrosine phosphorylation plays an important role in cell signaling and many physiological activities, which include tissue differentiation and growth, sugar and fat metabolism, and immune response [9,10]. Protein tyrosine phosphatase 1B (PTP1B), which is the first purified mammalian PTP, is selected as a reference prototype for later PTP studies . The precise regulation of protein tyrosine phosphorylation is determined by the concerted activities of PTKs and PTPs . The recombinant protein tyrosine kinase is indispensable as a target protein and model molecule in drug screening programs and its preparation is very important in the identification of anti-cancer therapeutics. Because the PTKs can phosphorylate intracellular proteins, disturb cellular homeostasis and result in toxicity to the host cells, the expression of recombinant PTK in P. pastoris cells becomes difficult . However, the successful co-expression of PTKs EGFR-2 and PDGFRβ with PTPs has been reported in our previous study .
In this work, the expression of c-Src by means of PTPs in P. pastoris was conducted to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells.
Material and Methods
Strains and culture conditions
The strains and plasmids used in this study are listed in Table 1. The yeast cells were grown in four different media at 30°C as described previously . Escherichia coli cells were routinely grown at 37°C in Luria-Bertani medium (LB; Oxoid, Basingstoke, UK). Antibiotics or other reagents were purchased from Sigma (St. Louis, MO, USA) and selectively added (750 μg ml-1 hygromycin, 50 μg ml-1 ampicillin, 50 μg ml-1 kanamycin, or 300 μg ml-1 G418).
Table 1. Strains and plasmids used in this study.
* SIMM, Shanghai Institute of Material Medica, Chinese Academy of Science.
Standard procedures for the manipulation of plasmid DNA were followed , and the plasmids and primers used for this study are listed in Table 1 and Table 2, respectively. The plasmids were constructed and transformed into P. pastoris according to our previous method .
Table 2. Primers used in this study.
a Restriction enzyme sites used for cloning of PCR products were underlined.
Construction of PTP1B expression strains
PTP1B expression strains 1Y, 2Y, 3Y and 4Y were constructed in our previous study .
Construction of c-Src expression strains
To construct PTK expression strains, the gene encoding c-Src was amplified from pBA3CS using primers S1/S2. The fragment was linked with SpeI/NotI-digested pPIC3.5K-His-GFP-SpeI-PDGFRβ-SKL-NotI plasmids to generate pPIC3.5K-Kozak- His10-linker-GFP-Linker-Thrombin site-c-Src (pPIC3.5-GFP-c-Src). This plasmid was confirmed by PCR with primers S1/S2 (Table 2) and DNA sequencing. The positive plasmid pPIC3.5-GFP-c-Src was linearized by either SalI or PmiI-digestion and electroporated into P. pastoris GS115 and 1Y~4Y to produce strains C and 1CP~4CP, respectively. The obtained five strains were listed in Table 1, and all of them were under the control of an AOX1 promoter. The His-deficient plates and YPD-G418 were used to screen the correct P. pastoris transformants. In order to further verify the strains, PCR amplification of strains C and 1CP~4CP was performed with primers S1/S2 and a product of 1648 bp was obtained, which was same as the theoretical value of c-Src.
Preparation of c-Src proteins
Strains 1CP, 2CP, 3CP, 4CP, and C were cultured in shaking flasks. The expression level of GFP in P. pastoris was determined by using flow cytometry (FACScalibur, Becton Dickinson, Franklin Lakes, NJ, USA). The cell extract was prepared as described previously . The concentration of protein was measured according to the method of Bradford (Tiangen Ltd., Shanghai, China). The expression level of c-Src was determined by using Western blotting .The c-Src protein was purified by his-tag affinity and ion-exchange chromatography, which were performed on the AKTA Explorer 100 purification system (Pharmacia Bioscience). The c-Src kinase activity and inhibition rate, using poly(Glu4-Tyr) as the substrate, were assayed by ELISA method with a tyrosine kinase assay kit (Millipore, USA).
Co-expression of BFP-PTP1B and GFP fused c-Src in P. pastoris
P. pastoris strains bearing the plasmids pPIC3.5-GFP-c-Src, pAG32S-BFP-PTP1B and pAG32S-BFP-PTP1B-SKL could express GFP, BFP and BFP-SKL which specifically targeted the peroxisome of P. pastoris . As expected, when BFP with GFP were co-expressed in P. pastoris strains 3CP and 4CP, the blue or green fluorescence was observed by fluorescence microscopy (Figure 1C, D). This result was the evidence that PTP1B can be co-expressed with c-Src in P. pastoris cells.
As shown in Figure 1D, both the blue fluorescence and the green fluorescence were nearly widespread in the entire cells in strain 4CP. The BFP-PTP1B was expressed in the cytoplasm without the carboxyl-terminal SKL. By contrast, a typical punctate blue fluorescence was observed in strain 3CP, which suggests that the fusion proteins BFP-PTP1B-SKL was sorted into the peroxisome with the SKL signal, whereas the green fluorescence was nearly widespread in the entire cells (Figure 1C). For control strain C containing pPIC3.5-GFP-c-Src (Figure 1E), strain 1CP containing PTP1B-SKL without BFP (Figure 1A) and strain 2CP containing PTP1B without BFP (Figure 1B), only green fluorescence was observed.
Figure 1. Fluorescence microscopy images of P. pastoris cellsexpressing different proteins. The cultures was induced toexpress the foriegn proteins with GFP and the cell pellets collected by centrifugation. The resuspended cells in PBS wereapplied to a slide, and fluorescence scatters of single cell were observed on a laser scanning confocal microscope (LSCM, LeicaDMRE) with an excitation wavelength of 488nm. (A) Strain 1CP co-expressing PTP1B-SKL and GFP-c-Src; (B) Strain 2CPco-expressing PTP1B and GFP-c-Src; (C) Strain 3CP co-expressing BFP-PTP1B-SKL and GFP-c-Src; (D) Strain 4CP co-expressingBFP-PTP1B and GFP-c-Src; (E) Strain C expressing GFP-c-Src. (a) differential interference contrast (DIC) image of cells; (b) cellsexpressing GFP protein; (c) cells expressing BFP-SKL protein in (C) or cells expressing BFP protein in (D) .
Expression, purification, and activity analysis of c-Srcproteins
Five strains C and 1CP~4CP, were cultured and inducedwith methanol in shaking flasks and sampled at 24hintervals.
The expression level of c-Src in each strain was determined according to the green fluorescence intensity of the fusedGFP protein to select out high yield strains. Under methanol induction, GFP was expressed in strains C and 1CP~4CP,and the maximum was achieved at 24h in strains 3CP and 4CP with a negligible difference (Table 3).
Table 3. Expression and tyrosine kinase activity of c-Src indifferent P. pastoris strains. Cell pellets grown in MGY mediumwere washed with water and then resuspended in MM medium to an OD600 of 1.0 for methanol induction for 48h in shake flasks.
a The Geo-Mean GFP fluorescence intensities were calculated with104 cells per analysis.
To support the utility of this expression system, we havepurified c-Src fusion proteins by using His-tag affinity andion-exchange chromatography. The c-Src fusion proteins in the cell lysate were determined to be 93 kDa in size bywestern blotting (Figure 2). The c-Src is a potential target for anti-cancer drug screening. The c-Src kinase activitywas measured using an ELISA method (Table 3, Figure 3A). The fraction of c-Src kinase activity was obtained from aHis-tag affinity column with a 250 mM imidazole elution. Moreover, the strain 3CP (Table 3) can obtain the highestkinase activity, which was consistent with the green fluorescence intensity in this strain. The BFP-PTP1B-SKLwas co-expressed with GFP-c-Src in strain 3CP, and the PTP1B activity was sorted in host peroxisome by targetingsignal SKL.
Figure 2. Western blot analysis of cell lysates (A) and their elute collections of Ni2+ affinity chromatography (B). Cell pellets grown in MM medium for 24h were disrupted and centrifugated, and the supernatants and their elute collections (electrophoretic purity) of Ni2+ affinity chromatography were measured by Western blot analysis using the GFP antibody. Lane 1: 1CP; Lane 2: 2CP; Lane 3: 3CP; Lane 4: 4CP strains expressing c-Src fusion protein in P. pastoris .
Tyrosine kinase substrate phosphorylation is a complex enzymatic process with double-substrate including poly (Glu4-Tyr) and ATP. However, the molecular model for protein tyrosine phosphatase screening c-Src inhibitors was investigated. The concentration of one substrate would be first determined, and that of another substrate could be then changed to demonstrate its relationship with the phosphorylation reaction. When the ATP was fixed, the substrate phosphorylation significantly increased with ATP concentration (data not shown), if the concentration of ATP was in a range of 1-10 μM. The optimal concentration of poly(Glu4-phospho-Tyr) and ATP was 10 ng well-1 and 5 μΜ, respectively. With an increase of c-Src, substrate phosphorylation increased rapidly and reached a plateau when c-Src was 48 ng well-1 (Figure 3). The tyrosine kinase activities in strains 1CP~4CP co-expressing with PTP1B were higher than that in strain C expressing the enzyme alone (Figure 3).
Figure 3. ELISA assay of the phosphorylation activity of c-Src. The relationship between phosphorylation states of substrate and c-Src quantity was illustrated by plot of A450 vs. c-Src quantity. In 96-well plates 10 ng well-1 poly(Glu4-Tyr) was pre-coated as a substrate. 85 μl of reaction buffer (100 mM Tris-HCl pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.5 mM Na3VO4, 0.5 mM DTT, 10 μM ATP) was added to each well. The reactions were performed with the addition of 15 μl purified c-Src fusion protein at varying concentrations .
c-Src is an important therapeutic target for drug discovery against cancer because of their critical role in osteoporosis. However, the inability to express active c-Src is associated with the c-Src kinase activity, which can phosphorylate intracellular proteins and result in toxicity to host cells . Our previous study demonstrates that the presence of PTP1B can overcome the toxicity of expressing an ac-tive PTK in P. pastoris, and provide practical strategies of recombinantly producing active PTKs in P. pastoris . Hence, c-Src was expressed by means of PTP1B in P. pastoris to overcome the potential negative effect caused by overexpression of c-Src. The in vivo activition of Src kinase is regulated by the phosphorylation status and some factors including protein tyrosine phosphatase 1B [18,19]. Bjorge et al.  found that protein-tyrosine phosphatase 1B was capable of dephosphorylating and activating c-Src in several human breast cancer cell lines. However, in our expression system, there was a balance between the dephosphorylation and the c-Src activation by PTP1B, and the dephosphorylation effect of PTP1B might be dominant with respect to the c-Src activation, which caused the balance to tilt to the dephosphorylation. Thus, enhance of c-Src expression could be expected.
P. pastoris usually utilizes the highly efficient methanolinduced AOX1 promoter (PAOX1) for high-level expression of foreign genes . To decrease the toxicity of PTP1B expression, phosphatase PTP1B with peroxisomal targeting signal 1 (SKL) was expressed and targeted to the peroxisome, which facilitates the separation of toxic metabolic products from the cell components . c-Src was expressed by means of phosphatase PTP1B with/without SKL in P. pastoris. The activity of c-Src co-expressed with PTP1B was compared to that without PTP1B in P. pastoris after growth in medium containing methanol. The peroxisomal targeting signal 1 (SKL) could influence the expression of c-Src. With SKL co-expression, GFP fused with c-Src was increased (Figure 2B, Lane 1&3). Except BFP, strain 3CP also included the signal peptide SKL, which could specifically target the GFP-c-Src fusion protein to the peroxisomes. Perhaps the location of GFP-c-Src was one of the reasons of higher expression in 3CP. However, enhanced GFP image in 4CP (Figure 2B, Lane 4) could not be reasonably clarified, and might be related with BFP expression in 4CP.
In conclusion, our results showed that the expression of c-Src by means of PTP1B in P. pastoris could increase the expression level of c-Src protein. Obviously, for c-Src expression, the kinase activities in strains 1CP~4CP were higher than that in strain C (Figure 3). The GFP fluorescence intensity reflected the expression of c-Src fusion protein. The highest intensities at 24h were achieved by strain 3CP and 4CP (Table 3), which was 62-67% higher than strain C. However, in strain 3CP, the tyrosine kinase activity of purified c-Src product in the elute collections of 250 mM imidazole was the highest among all the recombinant strains.
The kinase activity results indicated that expression of c-Src by means of PTP1B in P. pastoris can partly overcome the negative effects associated with overexpression of tyrosine kinases. Meanwhile, the tyrosine phosphatase PTP1B expression sorting at peroxisomes can reinforce the effect. A co-expression strategy has been developed which provides a valuable tool for the production of c-Src. This expression system interferes with the level of protein phosphorylation in host cells, and may prove to be useful for producing other recombinant PTKs as well.
This work was financially supported by National Special Fund for State Key Laboratory of Bioreactor Engineering (SKLBE-2013ZD010).