Examination of alternate codon bias solutions for expression and purification of recombinant mechano-growth factor in Escherichia coli
Jianguo Feng Rongxue Wan Qian Yi Ling He Li Yang ∗ Liling Tang
Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People’s Republic of China
Abstract Mechano-growth factor (MGF), an alternative splicing variant of insulin-like growth factor-1 (IGF-1) gene, promotes cell proliferation and inhibits cell differentiation. It also plays an important role in tumor development. It is important to optimize the production process and achieve MGF protein because there is no commercial MGF protein available. In this study, the human MGF gene is cloned into pGEX-4T-1 and the recombinant human MGF (rhMGF) protein could be expressed in Rosetta (DE3) by isopropyl β-D-1-thiogalactopyranoside induction but not in BL21 (DE3). Mutation from rare codons to Escherichia coli preferred ones is performed. We obtain MGF(Mut54–56) and MGF(Mut-total) fragments through site-directed mutagenesis and overlapping PCR. Both pGEX-4T-1/MGF(Mut54–56)- and pGEX-4T-1/
MGF(Mut-total)-transformed BL21 (DE3) can be induced to express rhMGF protein. To optimize the production technology, expression and purification of rhMGF are analyzed and compared in Rosetta (DE3) and BL21 (DE3). Results indicate that rhMGF expression in BL21 (DE3) is significantly higher than that in Rosetta (DE3). The protein yield of pGEX-4T-1/MGF(Mut-total) in BL21 (DE3) is higher than that of pGEX-4T-1/MGF(Mut54–56). We test the biological activity of MGF protein purified by affinity chromatography in C2C12 cell line and find that rhMGF promotes cell proliferation significantly. In conclusion, we establish a method to produce rhMGF economically with high biological activity in BL21 C 2014 International Union of Biochemistry and Molecular Biology, (DE3). Inc. Volume 62, Number 5, Pages 690–698, 2015
Keywords: mechano-growth factor, protein expression and purification, recombinant protein, site-directed mutagenesis
1. Introduction The insulin-like growth factor-1 (IGF-1) is a protein that in humans is encoded by the gene IGF-1 that spans for more than
Abbreviations: CCK-8, Cell Counting Kit-8; DMEM, Dulbecco’s minimum essential medium; DTT, dithiothreitol; IGF-1, insulin-like growth factor-1; IPTG, isopropyl-β-D-1-thiogalactopyranoside; MALDI-TOF, Matrix Assisted Laser Desorption Ionization Time-of-Flight; MGF, mechano-growth factor; MS, mass spectrum; PBS, phosphate buffered saline; rhMGF, recombinant human mechano-growth factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST, Tris-buffered saline plus 0.1% Tween 20.. ∗ Address for correspondence: Liling Tang, PhD, Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China. Tel.: +86 23 65102507; Fax: +86 23 65111901; e-mail: [email protected]. Supporting Information is available in the online issue at wileyonlinelibrary.com. Received 30 March 2014; accepted 20 October 2014
DOI: 10.1002/bab.1312 Published online 6 August 2015 in Wiley Online Library (wileyonlinelibrary.com)
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90 kb. IGF-1 has been shown to promote cell proliferation, differentiation, survival [1], and also to be involved in muscle maintenance, repair, and aging [2]. In humans, the IGF-1 gene generates three isoforms termed IGF-1Ea, IGF-1Eb, and IGF1Ec during tissue regeneration or in response to mechanical stimulation [2]. Mechano-growth factor (MGF) is one isoform of IGF-1, and named IGF-I Eb in rodents and corresponds to IGF-I Ec in humans [3]. MGF was first reported in rabbit skeletal muscle responding to mechanic stimulation [4]. Additionally, MGF is significantly increased in infarcted myocardium [5], the ischemic brain [6], osteoblasts following cyclic stretching [7], and tendon following eccentric training [8]. Recently, several studies reported that MGF was overexpressed in prostate cancer [9], and associated with prostate cancer stage [10]. Thus, MGF is probably an important factor in different cells and tissues. Actually, MGF has been tested as a potential therapy for muscular dystrophy and cerebral hypoxia-ischemia using experimental animals [11]. However, the functional mechanism of MGF is still poorly understood, which makes the harvesting of bioactive MGF protein more important.
Currently, there is no commercial human MGF available. In this study, we want to establish a method to produce recombinant human MGF (rhMGF). Escherichia coli, commonly used for economical production of recombinant proteins [12], serves as the host to produce rhMGF. Rosetta (DE3) and BL21 (DE3) are two E. coli strains, in which Rosetta (DE3) is a derivative of BL21 (DE3) and contains a plasmid encoding rare codon tRNAs including AUA, AGG, AGA, CUA, CCC, and GGA. Both Rosetta (DE3) and BL21 (DE3) are assessed in this study. rhMGF protein expression could be induced in Rosetta (DE3) but not in BL21 (DE3). With different site-directed mutagenesis, both MGF(Mut54–56) and MGF(Mut-total) could be induced to express protein in BL21 (DE3). Data show that the productivity of MGF(Mut54–56) and MGF(Mut-total) in BL21 (DE3) is significantly higher than that of MGF in Rosetta (DE3).
2. Materials and Methods 2.1. Recombinant plasmids cloning Full-length human MGF (AX147742) was amplified from pMD18-T/MGF (purchased from Invitrogen, USA) using primers MGF-F and MGF-R. The PCR product was gel purified and cloned into pGEX-4T-1 (Invitrogen) at BamHI and XhoI restriction enzyme sites. MGF(Mut54–56) fragment was amplified according to the procedure shown in Fig. 1A utilizing sitedirected mutagenesis PCR for changing three consecutive rare codons, coding the 54th–56th amino acids in MGF, into E. coli favorable ones, and then cloned into pGEX-4T-1. MGF(Muttotal) fragment was amplified according to the procedure shown in Fig. 1B utilizing site-directed mutagenesis PCR for changing all the rare codons in MGF into E. coli favorable ones, and then cloned into pGEX-4T-1. The details of the primers are summarized in Table 1.
2.2. rhMGF protein expression in different strains pGEX-4T-1/MGF construct was transformed into E. coli Rosetta (DE3) compentant cells (Beyotime, China). pGEX4T-1/MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) were transformed into E. coli BL21 (DE3) (Beyotime). Competent cells were plated onto LB agar plate containing 100 μg/mL ampicillin and cultured at 37 ◦ C overnight. Colonies of transformants were cultured in LB medium containing 100 μg/mL ampicillin at 37 ◦ C and 220 rpm until OD600 reached to 0.4–0.6. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was utilized to induce protein expression of rhMGF. Conditions for rhMGF proteins expression in E. coli Rosetta (DE3) were optimized, including IPTG concentration, induction time, and induction temperature. After the optimization was conducted, aliquots were analyzed by SDS-PAGE.
2.3. Purification of MGF protein The expression of human MGF was induced by IPTG at optimal conditions. Cell pellets were harvested by centrifugation. Subsequently, cell pellets were freeze–thawed for three times, and resuspended in ice-cold phosphate buffered saline (PBS) containing 1 mM DTT and protease inhibitor cocktail tablets
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(Roche, Switzerland). Cells were ultrasonicated and centrifuged at 8300g for 10 Min at 4 ◦ C. The supernatants were analyzed by SDS-PAGE. The supernatants were analyzed by Western blot to confirm that the target protein was obtained. The supernatants were separated by 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline plus 0.1% Tween 20 (TBST) and 5% nonfat dry milk. Then, membranes were incubated in anti-GST antibody (Bioworld, USA) diluted in 5% milk-TBST overnight at 4 ◦ C. After washed three times with TBST, membranes were incubated with a horseradish peroxidase conjugated anti-mouse-IgG antibody (ZSGB-BIO, China). Protein detection was performed using chemiluminescence (Millipore, USA) and VersaDoc Imaging system (Bio-Rad). Analysis of band intensity was performed using Quantity One image analysis software. Cell lysate was incubated with glutathione-sepharose beads (GE, USA) in a microcentrifuge tube at 4 ◦ C for1 H. Beads were washed with ice-cold PBS for three times. Elution buffer (50 mM reduced glutathione, PBS, 1 mM DTT) was incubated with the beads at room temperature (25 ◦ C) for approximately 30 Min. The eluted protein was analyzed by SDS-PAGE and Western blot.
2.4. MALDI-TOF MS The eluted protein was separated by SDS-PAGE. Then, the protein bond was excised and digested with trypsin and the main peptides were further analyzed to obtain the MS–MS spectrum using the ABI 4800 MALDI-TOF MS (Matrix-assisted laser desorption ionizationtime of flight mass spectrometry) spectrometer. The spectrum was analyzed using Mascot.
2.5. Protein biological activity assay MGF was regarded as a growth factor for myoblasts [13]. To determine the cellular proliferation activity of rhMGF, mouse C2C12 myoblasts cells (Cell Bank of the Chinese Academy of Medical Sciences) were grown in 96-well plates at a density of 1,000 cells per well in Dulbecco’s minimum essential medium (DMEM; Thermo), supplemented with 10% fetal bovine serum (Hyclone, USA), cultured for 12 H at 37 ◦ C in humid atmosphere containing 5% CO2 . rhMGF was diluted with DMEM supplemented with 2% fetal bovine serum to 1 and 5 nM, and then added to the plates. After 48 H of incubation, proliferation of C2C12 cells was analyzed by a Cell Counting Kit-8 (CCK-8, Beyotime) according to manufacturer’s instructions. The absorbance was assayed at 450 nm using a microplate reader (Bio-Rad). In the protein activity assay, GST was used as a negative control and IGF-1 (PeproTech) was tested as a positive control.
3. Results 3.1. Cloning of MGF and expression in Rosetta (DE3) and BL21 (DE3) Human MGF was cloned into expression vector pGEX-4T-1, which expressed GST fusion proteins. The sequencing result
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FIG. 1
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Strategies of site-directed mutagenesis. (A) MGF(Mut54–56) was obtained through site-directed mutation and overlapping PCR using MGF as the template. (B) MGF(Mut-total) was obtained through site-directed mutation and overlapping PCR using MGF(Mut54–56) as the template.
Expression and Purification of rhMGF in Two Escherichia coli Strains
Summary of primers
TABLE 1 MGF-F
5 -CGCGGATCC GGACCGGAGACGCTCTGC-3 (BamHI)
MGF-R
5 -’ CCGCTCGAGCTACTTGTGTTCTTCAAATGT-3 (XhoI)
MGF-Mut54–56 -F
5 -GATCTGCGTCGCCTGGAGATGTATTGCGCA-3
MGF-Mut54–56 -R
5 -CAGGCGACGCAGATC ACAGCTCCGGAAGCA-3
Mgf-1-R
5 -GCCATACCCTGTCGGCTTGTTGAAATAAAAGCCGCGGTCTCCACA-3
Mgf-2-F
5 -GGGTATGGCTCCAGCAGTCGGCGCGCGCCTCAG-3
Mgf-3-F
5 -TGCGCACCGCTCAAGCCTGCCAAG-3
Mgf-3-R
5 -CTTGGCAGGCTTGAGCGGTGCGCA-3
Mgf-4-R
5 -GGTAGATGGCGGCTGATACTTCTGGGTCTTCGGCATGTC-3
Mgf-5-R
5 -TTTGCGGCGCTGAGACTTCGTGTTCTTGTTGGTAGATGG-3
Mgf-6-R
5 -CTACTTGTGTTCTTCAAATGTACTTCCTTTGCGGCG-3
FIG. 2
Recombinant plasmids were induced in Rosetta (DE3) and BL21 (DE3). (A) pGEX-4T-1/MGF could be induced to express rhMGF in Rosetta (DE3) but not in BL21 (DE3). (B) Both pGEX-4T-1/ MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) were induced to express rhMGF in BL21 (DE3).
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FIG. 3
The DNA and amino acid sequence of MGF and IGF-1. (A) Results of RaCC analysis on MGF sequence. agg, aga = rare Arg codons; CTA = rare Leu codon; ccc = rare Pro codon. (B) The DNA sequence of MGF(Mut54–56). The letters in capitals are the mutant sites. (C) The DNA sequence of MGF(Mut-total). The letters in capitals are the mutant sites. (D) The amino acid sequence of mature IGF-1. (E) The amino acid sequence of MGF.
(Fig. S1 in the Supporting Information) showed the achievement of recombinant plasmid pGEX-4T-1/MGF. pGEX-4T-1/MGF was transformed into Rosetta (DE3) and BL21 (DE3), respectively. Fusion protein expression was induced for 5 H at 30 ◦ C with 0.4 mM IPTG. The results of SDS-PAGE (Fig. 2A) showed that rhMGF expression could be induced in Rosetta (DE3) but not in BL21 (DE3). Rosetta (DE3) is a strain derivative of BL21 and designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. Therefore, it is probable that the rare
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codons lead to rhMGF nonexpression in BL21 (DE3). The E. coli codon usage of human MGF gene was analyzed by the online software RACC (http://nihserver.mbi.ucla.edu/RACC/). The result of analysis (Fig. 3) showed that there were 11 rare codons in total, three consecutive rare codons included coding the 54th–56th amino acids.
3.2. Codon bias impedes expression of recombinant MGF in BL21 (DE3) It has been reported that rare codon clusters induce high levels of stalling and tagging by tRNA [14, 15]. Considering there are three consecutive rare codons in the human MGF gene, we changed the three rare codons coding the 54th–56th amino acids into E. coli preferred ones through site-directed mutagenesis PCR according to the procedure shown in Fig. 1A. Furthermore, the number of rare codons may influence the expression efficiency of target protein. We changed all the rare codons into E. coli preferred ones through PCR site-directed mutagenesis
Expression and Purification of rhMGF in Two Escherichia coli Strains
FIG. 4
Optimization of rhMGF expression conditions in Rosetta (DE3). (A) The effect of IPTG concentration on rhMGF expression in Rosetta (DE3). It has been found that 0.4 mM IPTG led to more protein expression. (B) The effect of induction time on rhMGF expression in Rosetta (DE3). The protein expression was greater at 5 H. (C) The effect of induction temperature on rhMGF expression in Rosetta (DE3). There were no significant differences among evaluated temperatures. Because of lower induction temperature leading to more soluble protein, we chose 16 ◦ C as the induction temperature for subsequent experiments.
according to the procedure shown in Fig. 1B. The sequencing results (Figs. S2 and S3 in the Supporting Information) showed the achievement of recombinant plasmids MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total). The DNA sequence of MGF(Mut54– 56) and pGEX-4T-1/MGF(Mut-total) are shown in Fig. 3. Two recombinant plasmids were transformed into BL21 (DE3), induced by IPTG, and analyzed by SDS-PAGE. The results (Fig. 2B) indicated that both recombinant plasmids could be expressed in BL21 (DE3). Our data confirmed that the rare codons in the human MGF gene impede the protein expression in BL21 (DE3).
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3.3. Optimization of rhMGF protein expression conditions in Rosetta (DE3) and protein purification The protein expression conditions, including IPTG concentration, induction time, and induction temperature, have a great influence on the productivity of recombinant proteins. Therefore, it is necessary to optimize the expression conditions of target proteins. The effects of conditions on the rhMGF expression in Rosetta (DE3) were evaluated, and the results are shown in Fig. 4. Data indicated that 0.4 mM IPTG and 5 H were the optimal conditions. In addition, the temperature ranging from 16 to 25 ◦ C was also evaluated, but the results (Fig. 4C) showed that there were no significant differences. Previous studies reported that low temperature improved the solubility of fusion proteins [16]. Therefore, we chose 16 ◦ C as the induction temperature for all the subsequent experiments. Cells were harvested by centrifuge and subsequently ultrasonicated. The supernatant was analyzed by SDS-PAGE (Fig. 5A) and Western blot (Fig. 5B). The supernatant was bound to glutathione-sepharose 4B, and then we washed the column with PBS to remove nonspecific proteins. The fusion protein was eluted by elution buffer. The eluted protein was analyzed by SDS-PAGE (Fig. 5A) and Western blot (Fig. 5B). The protein concentration was measured by the method using bovine serum albumin (Beyotime) as a standard. The final
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FIG. 5
rhMGF protein expression and purification in Rosetta (DE3). (A) The supernatant after ultrasonication and eluted protein were analyzed by SDS-PAGE. (B) The supernatant after ultrasonication and eluted protein were analyzed by Western blot.
yield of soluble rhMGF protein in Rosetta (DE3) averaged 1.33 mg/L of cell culture under the optimal induction conditions. The results are shown in Fig. 5, which indicated high yield of relatively specific target protein. The purified protein was also analyzed by the MALDI-TOF-TOF MS (Sangon Biotech, Fig. S4 in the Supporting Information) and probed with IGF-1 antibody (Proteintech, China) by Western blot (Fig. S5 in the Supporting Information). Moreover, the GST and GST-IGF-1 expressions were induced in E. coli through the production of rhMGF protein, and then the comparison of band sizes was conducted using SDS-PAGE and Western blot (Fig. S5 in the Supporting Information). These results showed that the purified protein was MGF.
3.4. Comparison between Rosetta (DE3) and BL21 (DE3) for rhMGF protein expression Previous data showed that both pGEX-4T-1/MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) could be induced to express
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FIG. 6
rhMGF protein was expressed in two strains after induction. (A) The rhMGF expression in supernatants was analyzed by SDS-PAGE. (B) The rhMGF expression in supernatants was analyzed by Western blot. (C) The quantity of protein was estimated by image density analysis software (Quantity One) and the data are shown as the mean ± SD, **P < 0.01.
rhMGF in BL21 (DE3). To pioneer the commercial production of this protein in E. coli, we compared the target protein productivity between Rosetta (DE3) and BL21 (DE3). All experiments were conducted with the optimal growth and protein expression conditions (0.4 mM IPTG, 16 ◦ C, 5 H). The cells in equal volume of culture were harvested and ultrasonicated. The supernatants were analyzed by SDS-PAGE (Fig. 6A) and Western blot (Fig. 6B). The quantity of protein in the gel was estimated by image density analysis software. Results showed that the rhMGF protein pGEX-4T-1/MGF(Mut54–56) induced to express in BL21 (DE3) increased by approximately 40% as compared with that of pGEX-4T-1/MGF induced to express
Expression and Purification of rhMGF in Two Escherichia coli Strains
FIG. 7
The biological activity of purified rhMGF. Cell proliferation in response to purified rhMGF was tested by CCK-8. rhMGF (1 and 5 nM) promoted the proliferation of C2C12 cells significantly. GST protein was used as a control. The biological activity of IGF-1 was tested as a positive control. The mean values are expressed as mean ± SD, **P < 0.01.
in Rosetta (DE3). In addition, the rhMGF protein pGEX-4T1/MGF(Mut-total) induced to express in BL21 (DE3) increased by approximately 50%, indicating that the less number of rare codons might lead to more productivity. The method to obtain rhMGF protein expressed in BL21 (DE3) through site-directed mutagenesis could get a higher productivity than rhMGF protein expressed in Rosetta (DE3).
3.5. Protein biological activity assay CCK-8 was commonly used for the determination of cell viability in cell proliferation [17, 18]. According to the previous study on biological activity of MGF [3], we tested the purified rhMGF biological activity by CCK-8. The results (Fig. 7) showed that both 1 and 5 nM rhMGF promoted the proliferation of C2C12 cells significantly similar to the effect of IGF-1, indicating highly biological activity of purified protein.
4. Discussion Heterologous protein expression in E. coli is strongly affected by codon bias [19]. In this study, we found that the rare codons in the human MGF gene prevented the protein expression in BL21 (DE3), suggesting that codon favorability of the host strain was an important factor for heterologous protein expression. pGEX-4T-1/MGF(Mut54–56) was capable of being induced in BL21 (DE3), indicating that the three consecutive rare codons (CTAAGGAGG) coding the 54th–56th amino acids (LeuArgArg) were the key barrier for protein expression. In addition, the rare codons content influenced the productivity of rhMGF because pGEX-4T-1/MGF(Mut-total) produced a higher level of protein than pGEX-4T-1/MGF(Mut54–56) in BL21 (DE3). Previous studies supported that positions of rare codons played an important role in target protein expression [20, 21]. By studying the effect of rare codon “AGG” on streptokinase expression, Thangadurai et al. [21] assumed that the specific
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position of rare codons in the downstream of the start codon modulated the protein expression. In this study, we made a similar conclusion. Figure 3 shows that CTALeu codon and two tandem AGGArg codons constituted the three consecutive rare codons, which were proved as the key barrier for protein expression in BL21 (DE3). Nevertheless, the AGGArg codon occurred in other positions of MGF gene. pGEX-4T1/MGF(Mut54–56) was capable of being induced to express protein in BL21 (DE3), indicating that the AGGArg codon of MGF gene might not become the barrier preventing protein expression. Besides, there was only one rare codon CTA Leu in the full-length human MGF gene. Therefore, the only CTALeu was regarded as the exact barrier for protein expression. However, when CTA was replaced with CTG, no protein expression was observed in BL21 (DE3) after induction (data not shown). It seemed that the position of rare codons, especially ones coding Arg, indeed played an important role in the protein expression, suggesting that the effect of distribution of rare codons should not be ignored in protein expression. The existence of rare codons in heterologous genes causes insufficient tRNA pools, leading to low levels of proteins expression. Thus, improving the abundance of tRNAs for rare codons in host cells is an effective way to get a higher level of proteins [22]. Rosetta (DE3), a derivative of BL21 (DE3), is a rare codon optimizer, and the effect of the two strains on the production of recombinant human protein was evaluated in Tegel et al.’s study [23]. Data showed that Rosetta (DE3) took a significant advantage for expression of human protein fragments mainly resulting from the content of rare codons. However, Rosetta (DE3) was not widely used in industrial production, probably due to its low growth rate and productivity. In this work, we observed that Rosetta (DE3) grew much more slowly than BL21 (DE3), which was a serious defect in industrial production for Rosetta (DE3). Based on our data, the protein yields of pGEX-4T-1/MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) in BL21 (DE3) are much higher than that of pGEX-4T-1/MGF in Rosetta (DE3). Therefore, the MGF(Mut-total) fragment with the highest protein yield in BL21 (DE3) could be potentially utilized for industrial production. Compared with IGF-1, MGF has a unique E domain at the C-terminus of protein in humans, leading to different functions of two proteins [24]. Here, we finally harvested the rhMGF proteins with GST tag, and the protein biological activity was tested on C2C12 cell line. Results indicated that we have obtained recombinant MGF with high biological activity. The research on different functions of IGF-1 and MGF will be conducted in the future work.
5. Conclusions In this work, human MGF gene could be induced to express protein in Rosetta (DE3) but not in BL21 (DE3) because of three consecutive rare codons (CTAAGGAGG) coding the 54th–56th amino acids (LeuArgArg). Two mutants, MGF(Mut54–56) and MGF(Mut-total), derived from human MGF gene were achieved through site-directed mutagenesis and overlapping PCR, and
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6. Acknowledgements This work was supported by the Natural Science Foundation of China (No. 31170890, 30970701), the “111” project (B06023, China), and the Fundamental Research Funds for the Central Universities (CDJXS112300). The authors declare no conflict of interest.
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Expression and Purification of rhMGF in Two Escherichia coli Strains
Jianguo Feng Rongxue Wan Qian Yi Ling He Li Yang ∗ Liling Tang
Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People’s Republic of China
Abstract Mechano-growth factor (MGF), an alternative splicing variant of insulin-like growth factor-1 (IGF-1) gene, promotes cell proliferation and inhibits cell differentiation. It also plays an important role in tumor development. It is important to optimize the production process and achieve MGF protein because there is no commercial MGF protein available. In this study, the human MGF gene is cloned into pGEX-4T-1 and the recombinant human MGF (rhMGF) protein could be expressed in Rosetta (DE3) by isopropyl β-D-1-thiogalactopyranoside induction but not in BL21 (DE3). Mutation from rare codons to Escherichia coli preferred ones is performed. We obtain MGF(Mut54–56) and MGF(Mut-total) fragments through site-directed mutagenesis and overlapping PCR. Both pGEX-4T-1/MGF(Mut54–56)- and pGEX-4T-1/
MGF(Mut-total)-transformed BL21 (DE3) can be induced to express rhMGF protein. To optimize the production technology, expression and purification of rhMGF are analyzed and compared in Rosetta (DE3) and BL21 (DE3). Results indicate that rhMGF expression in BL21 (DE3) is significantly higher than that in Rosetta (DE3). The protein yield of pGEX-4T-1/MGF(Mut-total) in BL21 (DE3) is higher than that of pGEX-4T-1/MGF(Mut54–56). We test the biological activity of MGF protein purified by affinity chromatography in C2C12 cell line and find that rhMGF promotes cell proliferation significantly. In conclusion, we establish a method to produce rhMGF economically with high biological activity in BL21 C 2014 International Union of Biochemistry and Molecular Biology, (DE3). Inc. Volume 62, Number 5, Pages 690–698, 2015
Keywords: mechano-growth factor, protein expression and purification, recombinant protein, site-directed mutagenesis
1. Introduction The insulin-like growth factor-1 (IGF-1) is a protein that in humans is encoded by the gene IGF-1 that spans for more than
Abbreviations: CCK-8, Cell Counting Kit-8; DMEM, Dulbecco’s minimum essential medium; DTT, dithiothreitol; IGF-1, insulin-like growth factor-1; IPTG, isopropyl-β-D-1-thiogalactopyranoside; MALDI-TOF, Matrix Assisted Laser Desorption Ionization Time-of-Flight; MGF, mechano-growth factor; MS, mass spectrum; PBS, phosphate buffered saline; rhMGF, recombinant human mechano-growth factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST, Tris-buffered saline plus 0.1% Tween 20.. ∗ Address for correspondence: Liling Tang, PhD, Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China. Tel.: +86 23 65102507; Fax: +86 23 65111901; e-mail: [email protected]. Supporting Information is available in the online issue at wileyonlinelibrary.com. Received 30 March 2014; accepted 20 October 2014
DOI: 10.1002/bab.1312 Published online 6 August 2015 in Wiley Online Library (wileyonlinelibrary.com)
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90 kb. IGF-1 has been shown to promote cell proliferation, differentiation, survival [1], and also to be involved in muscle maintenance, repair, and aging [2]. In humans, the IGF-1 gene generates three isoforms termed IGF-1Ea, IGF-1Eb, and IGF1Ec during tissue regeneration or in response to mechanical stimulation [2]. Mechano-growth factor (MGF) is one isoform of IGF-1, and named IGF-I Eb in rodents and corresponds to IGF-I Ec in humans [3]. MGF was first reported in rabbit skeletal muscle responding to mechanic stimulation [4]. Additionally, MGF is significantly increased in infarcted myocardium [5], the ischemic brain [6], osteoblasts following cyclic stretching [7], and tendon following eccentric training [8]. Recently, several studies reported that MGF was overexpressed in prostate cancer [9], and associated with prostate cancer stage [10]. Thus, MGF is probably an important factor in different cells and tissues. Actually, MGF has been tested as a potential therapy for muscular dystrophy and cerebral hypoxia-ischemia using experimental animals [11]. However, the functional mechanism of MGF is still poorly understood, which makes the harvesting of bioactive MGF protein more important.
Currently, there is no commercial human MGF available. In this study, we want to establish a method to produce recombinant human MGF (rhMGF). Escherichia coli, commonly used for economical production of recombinant proteins [12], serves as the host to produce rhMGF. Rosetta (DE3) and BL21 (DE3) are two E. coli strains, in which Rosetta (DE3) is a derivative of BL21 (DE3) and contains a plasmid encoding rare codon tRNAs including AUA, AGG, AGA, CUA, CCC, and GGA. Both Rosetta (DE3) and BL21 (DE3) are assessed in this study. rhMGF protein expression could be induced in Rosetta (DE3) but not in BL21 (DE3). With different site-directed mutagenesis, both MGF(Mut54–56) and MGF(Mut-total) could be induced to express protein in BL21 (DE3). Data show that the productivity of MGF(Mut54–56) and MGF(Mut-total) in BL21 (DE3) is significantly higher than that of MGF in Rosetta (DE3).
2. Materials and Methods 2.1. Recombinant plasmids cloning Full-length human MGF (AX147742) was amplified from pMD18-T/MGF (purchased from Invitrogen, USA) using primers MGF-F and MGF-R. The PCR product was gel purified and cloned into pGEX-4T-1 (Invitrogen) at BamHI and XhoI restriction enzyme sites. MGF(Mut54–56) fragment was amplified according to the procedure shown in Fig. 1A utilizing sitedirected mutagenesis PCR for changing three consecutive rare codons, coding the 54th–56th amino acids in MGF, into E. coli favorable ones, and then cloned into pGEX-4T-1. MGF(Muttotal) fragment was amplified according to the procedure shown in Fig. 1B utilizing site-directed mutagenesis PCR for changing all the rare codons in MGF into E. coli favorable ones, and then cloned into pGEX-4T-1. The details of the primers are summarized in Table 1.
2.2. rhMGF protein expression in different strains pGEX-4T-1/MGF construct was transformed into E. coli Rosetta (DE3) compentant cells (Beyotime, China). pGEX4T-1/MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) were transformed into E. coli BL21 (DE3) (Beyotime). Competent cells were plated onto LB agar plate containing 100 μg/mL ampicillin and cultured at 37 ◦ C overnight. Colonies of transformants were cultured in LB medium containing 100 μg/mL ampicillin at 37 ◦ C and 220 rpm until OD600 reached to 0.4–0.6. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was utilized to induce protein expression of rhMGF. Conditions for rhMGF proteins expression in E. coli Rosetta (DE3) were optimized, including IPTG concentration, induction time, and induction temperature. After the optimization was conducted, aliquots were analyzed by SDS-PAGE.
2.3. Purification of MGF protein The expression of human MGF was induced by IPTG at optimal conditions. Cell pellets were harvested by centrifugation. Subsequently, cell pellets were freeze–thawed for three times, and resuspended in ice-cold phosphate buffered saline (PBS) containing 1 mM DTT and protease inhibitor cocktail tablets
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(Roche, Switzerland). Cells were ultrasonicated and centrifuged at 8300g for 10 Min at 4 ◦ C. The supernatants were analyzed by SDS-PAGE. The supernatants were analyzed by Western blot to confirm that the target protein was obtained. The supernatants were separated by 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline plus 0.1% Tween 20 (TBST) and 5% nonfat dry milk. Then, membranes were incubated in anti-GST antibody (Bioworld, USA) diluted in 5% milk-TBST overnight at 4 ◦ C. After washed three times with TBST, membranes were incubated with a horseradish peroxidase conjugated anti-mouse-IgG antibody (ZSGB-BIO, China). Protein detection was performed using chemiluminescence (Millipore, USA) and VersaDoc Imaging system (Bio-Rad). Analysis of band intensity was performed using Quantity One image analysis software. Cell lysate was incubated with glutathione-sepharose beads (GE, USA) in a microcentrifuge tube at 4 ◦ C for1 H. Beads were washed with ice-cold PBS for three times. Elution buffer (50 mM reduced glutathione, PBS, 1 mM DTT) was incubated with the beads at room temperature (25 ◦ C) for approximately 30 Min. The eluted protein was analyzed by SDS-PAGE and Western blot.
2.4. MALDI-TOF MS The eluted protein was separated by SDS-PAGE. Then, the protein bond was excised and digested with trypsin and the main peptides were further analyzed to obtain the MS–MS spectrum using the ABI 4800 MALDI-TOF MS (Matrix-assisted laser desorption ionizationtime of flight mass spectrometry) spectrometer. The spectrum was analyzed using Mascot.
2.5. Protein biological activity assay MGF was regarded as a growth factor for myoblasts [13]. To determine the cellular proliferation activity of rhMGF, mouse C2C12 myoblasts cells (Cell Bank of the Chinese Academy of Medical Sciences) were grown in 96-well plates at a density of 1,000 cells per well in Dulbecco’s minimum essential medium (DMEM; Thermo), supplemented with 10% fetal bovine serum (Hyclone, USA), cultured for 12 H at 37 ◦ C in humid atmosphere containing 5% CO2 . rhMGF was diluted with DMEM supplemented with 2% fetal bovine serum to 1 and 5 nM, and then added to the plates. After 48 H of incubation, proliferation of C2C12 cells was analyzed by a Cell Counting Kit-8 (CCK-8, Beyotime) according to manufacturer’s instructions. The absorbance was assayed at 450 nm using a microplate reader (Bio-Rad). In the protein activity assay, GST was used as a negative control and IGF-1 (PeproTech) was tested as a positive control.
3. Results 3.1. Cloning of MGF and expression in Rosetta (DE3) and BL21 (DE3) Human MGF was cloned into expression vector pGEX-4T-1, which expressed GST fusion proteins. The sequencing result
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FIG. 1
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Strategies of site-directed mutagenesis. (A) MGF(Mut54–56) was obtained through site-directed mutation and overlapping PCR using MGF as the template. (B) MGF(Mut-total) was obtained through site-directed mutation and overlapping PCR using MGF(Mut54–56) as the template.
Expression and Purification of rhMGF in Two Escherichia coli Strains
Summary of primers
TABLE 1 MGF-F
5 -CGCGGATCC GGACCGGAGACGCTCTGC-3 (BamHI)
MGF-R
5 -’ CCGCTCGAGCTACTTGTGTTCTTCAAATGT-3 (XhoI)
MGF-Mut54–56 -F
5 -GATCTGCGTCGCCTGGAGATGTATTGCGCA-3
MGF-Mut54–56 -R
5 -CAGGCGACGCAGATC ACAGCTCCGGAAGCA-3
Mgf-1-R
5 -GCCATACCCTGTCGGCTTGTTGAAATAAAAGCCGCGGTCTCCACA-3
Mgf-2-F
5 -GGGTATGGCTCCAGCAGTCGGCGCGCGCCTCAG-3
Mgf-3-F
5 -TGCGCACCGCTCAAGCCTGCCAAG-3
Mgf-3-R
5 -CTTGGCAGGCTTGAGCGGTGCGCA-3
Mgf-4-R
5 -GGTAGATGGCGGCTGATACTTCTGGGTCTTCGGCATGTC-3
Mgf-5-R
5 -TTTGCGGCGCTGAGACTTCGTGTTCTTGTTGGTAGATGG-3
Mgf-6-R
5 -CTACTTGTGTTCTTCAAATGTACTTCCTTTGCGGCG-3
FIG. 2
Recombinant plasmids were induced in Rosetta (DE3) and BL21 (DE3). (A) pGEX-4T-1/MGF could be induced to express rhMGF in Rosetta (DE3) but not in BL21 (DE3). (B) Both pGEX-4T-1/ MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) were induced to express rhMGF in BL21 (DE3).
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FIG. 3
The DNA and amino acid sequence of MGF and IGF-1. (A) Results of RaCC analysis on MGF sequence. agg, aga = rare Arg codons; CTA = rare Leu codon; ccc = rare Pro codon. (B) The DNA sequence of MGF(Mut54–56). The letters in capitals are the mutant sites. (C) The DNA sequence of MGF(Mut-total). The letters in capitals are the mutant sites. (D) The amino acid sequence of mature IGF-1. (E) The amino acid sequence of MGF.
(Fig. S1 in the Supporting Information) showed the achievement of recombinant plasmid pGEX-4T-1/MGF. pGEX-4T-1/MGF was transformed into Rosetta (DE3) and BL21 (DE3), respectively. Fusion protein expression was induced for 5 H at 30 ◦ C with 0.4 mM IPTG. The results of SDS-PAGE (Fig. 2A) showed that rhMGF expression could be induced in Rosetta (DE3) but not in BL21 (DE3). Rosetta (DE3) is a strain derivative of BL21 and designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. Therefore, it is probable that the rare
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codons lead to rhMGF nonexpression in BL21 (DE3). The E. coli codon usage of human MGF gene was analyzed by the online software RACC (http://nihserver.mbi.ucla.edu/RACC/). The result of analysis (Fig. 3) showed that there were 11 rare codons in total, three consecutive rare codons included coding the 54th–56th amino acids.
3.2. Codon bias impedes expression of recombinant MGF in BL21 (DE3) It has been reported that rare codon clusters induce high levels of stalling and tagging by tRNA [14, 15]. Considering there are three consecutive rare codons in the human MGF gene, we changed the three rare codons coding the 54th–56th amino acids into E. coli preferred ones through site-directed mutagenesis PCR according to the procedure shown in Fig. 1A. Furthermore, the number of rare codons may influence the expression efficiency of target protein. We changed all the rare codons into E. coli preferred ones through PCR site-directed mutagenesis
Expression and Purification of rhMGF in Two Escherichia coli Strains
FIG. 4
Optimization of rhMGF expression conditions in Rosetta (DE3). (A) The effect of IPTG concentration on rhMGF expression in Rosetta (DE3). It has been found that 0.4 mM IPTG led to more protein expression. (B) The effect of induction time on rhMGF expression in Rosetta (DE3). The protein expression was greater at 5 H. (C) The effect of induction temperature on rhMGF expression in Rosetta (DE3). There were no significant differences among evaluated temperatures. Because of lower induction temperature leading to more soluble protein, we chose 16 ◦ C as the induction temperature for subsequent experiments.
according to the procedure shown in Fig. 1B. The sequencing results (Figs. S2 and S3 in the Supporting Information) showed the achievement of recombinant plasmids MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total). The DNA sequence of MGF(Mut54– 56) and pGEX-4T-1/MGF(Mut-total) are shown in Fig. 3. Two recombinant plasmids were transformed into BL21 (DE3), induced by IPTG, and analyzed by SDS-PAGE. The results (Fig. 2B) indicated that both recombinant plasmids could be expressed in BL21 (DE3). Our data confirmed that the rare codons in the human MGF gene impede the protein expression in BL21 (DE3).
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3.3. Optimization of rhMGF protein expression conditions in Rosetta (DE3) and protein purification The protein expression conditions, including IPTG concentration, induction time, and induction temperature, have a great influence on the productivity of recombinant proteins. Therefore, it is necessary to optimize the expression conditions of target proteins. The effects of conditions on the rhMGF expression in Rosetta (DE3) were evaluated, and the results are shown in Fig. 4. Data indicated that 0.4 mM IPTG and 5 H were the optimal conditions. In addition, the temperature ranging from 16 to 25 ◦ C was also evaluated, but the results (Fig. 4C) showed that there were no significant differences. Previous studies reported that low temperature improved the solubility of fusion proteins [16]. Therefore, we chose 16 ◦ C as the induction temperature for all the subsequent experiments. Cells were harvested by centrifuge and subsequently ultrasonicated. The supernatant was analyzed by SDS-PAGE (Fig. 5A) and Western blot (Fig. 5B). The supernatant was bound to glutathione-sepharose 4B, and then we washed the column with PBS to remove nonspecific proteins. The fusion protein was eluted by elution buffer. The eluted protein was analyzed by SDS-PAGE (Fig. 5A) and Western blot (Fig. 5B). The protein concentration was measured by the method using bovine serum albumin (Beyotime) as a standard. The final
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FIG. 5
rhMGF protein expression and purification in Rosetta (DE3). (A) The supernatant after ultrasonication and eluted protein were analyzed by SDS-PAGE. (B) The supernatant after ultrasonication and eluted protein were analyzed by Western blot.
yield of soluble rhMGF protein in Rosetta (DE3) averaged 1.33 mg/L of cell culture under the optimal induction conditions. The results are shown in Fig. 5, which indicated high yield of relatively specific target protein. The purified protein was also analyzed by the MALDI-TOF-TOF MS (Sangon Biotech, Fig. S4 in the Supporting Information) and probed with IGF-1 antibody (Proteintech, China) by Western blot (Fig. S5 in the Supporting Information). Moreover, the GST and GST-IGF-1 expressions were induced in E. coli through the production of rhMGF protein, and then the comparison of band sizes was conducted using SDS-PAGE and Western blot (Fig. S5 in the Supporting Information). These results showed that the purified protein was MGF.
3.4. Comparison between Rosetta (DE3) and BL21 (DE3) for rhMGF protein expression Previous data showed that both pGEX-4T-1/MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) could be induced to express
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FIG. 6
rhMGF protein was expressed in two strains after induction. (A) The rhMGF expression in supernatants was analyzed by SDS-PAGE. (B) The rhMGF expression in supernatants was analyzed by Western blot. (C) The quantity of protein was estimated by image density analysis software (Quantity One) and the data are shown as the mean ± SD, **P < 0.01.
rhMGF in BL21 (DE3). To pioneer the commercial production of this protein in E. coli, we compared the target protein productivity between Rosetta (DE3) and BL21 (DE3). All experiments were conducted with the optimal growth and protein expression conditions (0.4 mM IPTG, 16 ◦ C, 5 H). The cells in equal volume of culture were harvested and ultrasonicated. The supernatants were analyzed by SDS-PAGE (Fig. 6A) and Western blot (Fig. 6B). The quantity of protein in the gel was estimated by image density analysis software. Results showed that the rhMGF protein pGEX-4T-1/MGF(Mut54–56) induced to express in BL21 (DE3) increased by approximately 40% as compared with that of pGEX-4T-1/MGF induced to express
Expression and Purification of rhMGF in Two Escherichia coli Strains
FIG. 7
The biological activity of purified rhMGF. Cell proliferation in response to purified rhMGF was tested by CCK-8. rhMGF (1 and 5 nM) promoted the proliferation of C2C12 cells significantly. GST protein was used as a control. The biological activity of IGF-1 was tested as a positive control. The mean values are expressed as mean ± SD, **P < 0.01.
in Rosetta (DE3). In addition, the rhMGF protein pGEX-4T1/MGF(Mut-total) induced to express in BL21 (DE3) increased by approximately 50%, indicating that the less number of rare codons might lead to more productivity. The method to obtain rhMGF protein expressed in BL21 (DE3) through site-directed mutagenesis could get a higher productivity than rhMGF protein expressed in Rosetta (DE3).
3.5. Protein biological activity assay CCK-8 was commonly used for the determination of cell viability in cell proliferation [17, 18]. According to the previous study on biological activity of MGF [3], we tested the purified rhMGF biological activity by CCK-8. The results (Fig. 7) showed that both 1 and 5 nM rhMGF promoted the proliferation of C2C12 cells significantly similar to the effect of IGF-1, indicating highly biological activity of purified protein.
4. Discussion Heterologous protein expression in E. coli is strongly affected by codon bias [19]. In this study, we found that the rare codons in the human MGF gene prevented the protein expression in BL21 (DE3), suggesting that codon favorability of the host strain was an important factor for heterologous protein expression. pGEX-4T-1/MGF(Mut54–56) was capable of being induced in BL21 (DE3), indicating that the three consecutive rare codons (CTAAGGAGG) coding the 54th–56th amino acids (LeuArgArg) were the key barrier for protein expression. In addition, the rare codons content influenced the productivity of rhMGF because pGEX-4T-1/MGF(Mut-total) produced a higher level of protein than pGEX-4T-1/MGF(Mut54–56) in BL21 (DE3). Previous studies supported that positions of rare codons played an important role in target protein expression [20, 21]. By studying the effect of rare codon “AGG” on streptokinase expression, Thangadurai et al. [21] assumed that the specific
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position of rare codons in the downstream of the start codon modulated the protein expression. In this study, we made a similar conclusion. Figure 3 shows that CTALeu codon and two tandem AGGArg codons constituted the three consecutive rare codons, which were proved as the key barrier for protein expression in BL21 (DE3). Nevertheless, the AGGArg codon occurred in other positions of MGF gene. pGEX-4T1/MGF(Mut54–56) was capable of being induced to express protein in BL21 (DE3), indicating that the AGGArg codon of MGF gene might not become the barrier preventing protein expression. Besides, there was only one rare codon CTA Leu in the full-length human MGF gene. Therefore, the only CTALeu was regarded as the exact barrier for protein expression. However, when CTA was replaced with CTG, no protein expression was observed in BL21 (DE3) after induction (data not shown). It seemed that the position of rare codons, especially ones coding Arg, indeed played an important role in the protein expression, suggesting that the effect of distribution of rare codons should not be ignored in protein expression. The existence of rare codons in heterologous genes causes insufficient tRNA pools, leading to low levels of proteins expression. Thus, improving the abundance of tRNAs for rare codons in host cells is an effective way to get a higher level of proteins [22]. Rosetta (DE3), a derivative of BL21 (DE3), is a rare codon optimizer, and the effect of the two strains on the production of recombinant human protein was evaluated in Tegel et al.’s study [23]. Data showed that Rosetta (DE3) took a significant advantage for expression of human protein fragments mainly resulting from the content of rare codons. However, Rosetta (DE3) was not widely used in industrial production, probably due to its low growth rate and productivity. In this work, we observed that Rosetta (DE3) grew much more slowly than BL21 (DE3), which was a serious defect in industrial production for Rosetta (DE3). Based on our data, the protein yields of pGEX-4T-1/MGF(Mut54–56) and pGEX-4T-1/MGF(Mut-total) in BL21 (DE3) are much higher than that of pGEX-4T-1/MGF in Rosetta (DE3). Therefore, the MGF(Mut-total) fragment with the highest protein yield in BL21 (DE3) could be potentially utilized for industrial production. Compared with IGF-1, MGF has a unique E domain at the C-terminus of protein in humans, leading to different functions of two proteins [24]. Here, we finally harvested the rhMGF proteins with GST tag, and the protein biological activity was tested on C2C12 cell line. Results indicated that we have obtained recombinant MGF with high biological activity. The research on different functions of IGF-1 and MGF will be conducted in the future work.
5. Conclusions In this work, human MGF gene could be induced to express protein in Rosetta (DE3) but not in BL21 (DE3) because of three consecutive rare codons (CTAAGGAGG) coding the 54th–56th amino acids (LeuArgArg). Two mutants, MGF(Mut54–56) and MGF(Mut-total), derived from human MGF gene were achieved through site-directed mutagenesis and overlapping PCR, and
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6. Acknowledgements This work was supported by the Natural Science Foundation of China (No. 31170890, 30970701), the “111” project (B06023, China), and the Fundamental Research Funds for the Central Universities (CDJXS112300). The authors declare no conflict of interest.
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Expression and Purification of rhMGF in Two Escherichia coli Strains
