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Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli
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Q1
Tonglin Shi a, Lichao Zhang a, Zhuoyu Li a,b,⇑, Ian P. Newton c, Quanbin Zhang d a
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi 030006, China College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China c Cell and Developmental Biology, University of Dundee, Dundee, Scotland DD1 5EH, UK d Central Laboratory of Taiyuan Central Hospital, No. 1, East Sandao Alley, Xinghualing District, Taiyuan, Shanxi 030009, China b
a r t i c l e
i n f o
Article history: Received 22 September 2014 and in revised form 12 November 2014 Available online xxxx Keywords: Prokaryotic expression Integrin b1 pET-28a vector Inclusion body
a b s t r a c t Integrins are a family of transmembrane receptors and among their members, integrin b1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin b1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin b1. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells and after induction by isopropyl-b-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8 M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS–PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin b. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn2+ stimulation. Ó 2014 Published by Elsevier Inc.
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Introduction
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The integrin family consists of 24 members [1]. Their expression in cells, when triggered, relates to various functions of embryonic development, angiogenesis, cell proliferation, adhesion and migration etc [2,3]. Among these, integrin b1 is one of the most studied subunits. Integrin b1 is expressed in a cell- and tissue-specific manner, but almost universally expressed in tumor cells [4]. Its high expression results in the progression of some types of cancers [5]. Moreover, a2b1 [6], a3b1 [7,8], a5b1 [9,10], or a6b1 [11] have been identified as predictors for cancer metastasis. Integrins exist in two distinct conformations, one of which is ‘‘closed’’ (bent) and the other ‘‘open’’. These correspond to the inactive and activated forms of integrin, respectively [12]. How these two conformations switch is still unknown although research suggests that the relationship between the ligands and integrin
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⇑ Corresponding author at: Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006, China. Tel.: +86 351 7018268; fax: +86 351 7018268. E-mail address: [email protected] (Z. Li).
activation [12], or the signal transduction involved in this process [13] are critical. Being able to obtain enough purified integrin b1 protein is a prerequisite to carrying out these types of studies, especially for in vitro experiments. There have been a few studies focusing on the expression of the extracelluar region of integrin b1 [14], and to the best of our knowledge, none of them utilized a prokaryotic expression system. In present study, an efficient process was shown to produce highly purified, soluble and active integrin b1 ectodomain using Escherichia coli. A gene segment that represented 454 amino acids of the N-terminal of integrin b1 was cloned into pET-28a-c (+) (abbreviated as pET-28a) vector and transformed into E. coli BL21 (DE3) cells. The recombinant polypeptides (53 kD) were expressed as inclusion bodies. The inclusion bodies were purified by nickel affinity resin and refolded through stepwise dialysis. The refolded protein was successfully dissolved in phosphate buffered saline (PBS)1, and the yield was about 5.4 mg/L of culture. We proved that Q2
1 Abbreviations used: PBS, phosphate buffered saline; FBS, fetal bovine serum; IPTG, isopropyl-b-D-thiogalactopyranoside; PVDF, polyvinylidene difluoride.
http://dx.doi.org/10.1016/j.pep.2014.11.007 1046-5928/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: T. Shi et al., Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.007
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the renatured polypeptides had native structure and the correct bioactivities.
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Materials and methods
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Cell culture and cDNA synthesis
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NCM460 normal human colon mucosa epithelial cells were cultured in DMEM/F12 (1:1) culture medium (Hyclone) plus 10% fetal bovine serum (FBS, Sangon biotech, Shanghai, China), and grown in humidified air with 5% CO2. Cells were harvested using a cell scraper. Total RNA was isolated using RNAiso Plus (Takara Biotechnology) and purified according to the standard protocol. The cDNA was synthesized using PrimeScript 1st strand cDNA synthesis kit (Takara) according to the user manual.
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Construction of recombinant plasmid
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The PCR primers used for cloning the ectodomain of integrin b1 were designed (Table 1) according to the protein sequence included in PDB: 3VI3 (chain B) [15]. Restriction sites of Bam HI and Xho I were separately introduced to the forward or reverse primers in order to facilitate downstream operation. The specific target DNA segment was amplified on a thermocycler (Mastercycler personal, Eppendorf) using the following program: denaturation at 98 °C for 5 min, followed by 98 °C for 10 s, 15 s at 55 °C, 72 °C for 2 min for 35 cycles. Products underwent a final extension at 72 °C for 10 min. PrimeSTAR HS DNA Polymerase (Takara) was used to ensure high fidelity and accuracy during amplification. The PCR products were resolved using 1% agarose gel and extracted. Purified segments were ligated into pEASY-Blunt Zero Cloning vector (Transgen, Beijing, China) and transformed into Trans5a competent cells (Transgen). Cells were spread on LB plates containing 0.05 mg/mL kanamycin and cultured at 37 °C overnight. Positive clone was selected and grown in 5 mL LB liquid medium containing kanamycin. The recombinant plasmid DNA was extracted (Sangon, Shanghai, China) and digested using Bam HI and Xho I in buffer K (Takara) at 30 °C for 2 h. After gel extraction and purification, inserts were ligated into pET-28a vector (Novagen), that had been digested with the same restriction enzymes as mentioned above using DNA Ligation Kit Ver. 2.1 (Takara) with a ratio of insert: vector = 10:1 (mol/mol) as per the user manual. Recombinant pET-28a was transformed into BL21 (DE3) E. coli cells. Cells were grown overnight at 37 °C on LB plates with kanamycin. Positive colonies were identified by colony PCR, restriction digestion, and verified by DNA sequencing (Sangon, Shanghai, China).
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Expression of recombinant protein
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Positive colony was selected and used to inoculate 1 L of LB medium containing 50 lg/mL kanamycin. Cells were grown in a shaker at 37 °C until OD600 reached 0.9. Following induction with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) being added into the culture medium for 3 h at 37 °C with 200 rpm shaking, cells were harvested by centrifugation at 8000g, 4 °C for 10 min
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Table 1 Primers used for cloning the coding sequence of integrin b1 ectodomain. Underlined characters indicated the restriction sites, italic characters represented the termination signal of transcription. Primer name
Sequences
Restriction enzyme
ITGB1-head-F
50 -CGC GGA TCC CAA ACA GAT GAA AAT AGA TG
Bam HI
ITGB1-head-R
50 -CCG CTC GAG TTA TTC ACA TTC ACA GAT GTA C
Xho I
and washed with cold phosphate buffered saline (PBS, 0.2 g/L KCl, 3.63 g/L Na2HPO4-12H2O, 0.24 g/L KH2PO4, and 8 g/L NaCl, pH = 8.0). The pellets were resuspended in lysis buffer (containing 5 mM b-mercaptoethanol, 1% (w/v) sodium deoxycholate and 1 mM PMSF in PBS, pH = 8.0) then lysed using ultrasound. Intracellular compounds were isolated by 15,000g centrifugation at 4 °C for 15 min. Pellets were washed three times in wash buffer (2 M urea, 1% (v/v) Triton X-100 in PBS, pH = 8.0), then resuspended in extraction buffer (8 M urea, 0.5 M NaCl, 20 mM imidazole, 5 mM b-mercaptoethanol in PBS, pH = 8.0) and allowed to dissolve overnight at 4 °C.
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ITGB1-head purification and refolding
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Extracted inclusion bodies were centrifuged at 4500g (swingbucket rotor, Eppendorf) for 15 min to remove any insoluble debris. The supernatant was transferred to a new falcon tube. Protein concentration was assessed using the BCA method (Beyotime Institute of Biotechnology, Jiangsu, China), and was regulated to be lower than 0.2 mg/mL by extraction buffer to avoid protein aggregation. The protein solution was mixed with binding buffer (same as the above extraction buffer)-equilibrated Ni Sepharose 6 fast flow (GE healthcare) slurry and shaken gently for 1 h at room temperature. After centrifugation at 800g for 5 min, pellets were mixed with binding buffer at a ratio of 1:1 (v/v). The slurry was loaded on a PD-10 column (GE healthcare). Bound fractions were washed and eluted under denaturing conditions according to manufacturer’s instructions. The purified recombinant polypeptide was stepwise dialyzed at 4 °C with the following refolding buffers: (1) refolding buffer A (4 M urea, 0.1 mM glutathione, 0.01 mM glutathione disulfide, 1 mM EDTA, 5% (v/v) glycerol in PBS, pH = 8.0) for 12 h; (2) refolding buffer B (2 M urea, 0.1 mM glutathione, 0.01 mM glutathione disulfide, 1 mM EDTA, 5% (v/v) glycerol, 0.15 M L-arginine in PBS, pH = 8.0) for 12 h; (3) refolding buffer C (1 M urea, 0.1 mM glutathione, 0.01 mM glutathione disulfide, 1 mM EDTA, 5% (v/v) glycerol, in PBS, pH = 8.0) for 12 h; and (4) refolding buffer D (PBS, pH = 8.0) for 12 h, twice. Concentration of refolded ITGB1-head was determined by BCA protein assay kit.
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Purity and bioactivity assay
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The purity of recombinant ITGB1-head polypeptide was determined by SDS–PAGE. Briefly, 7.5 lg of each refolded protein sample was boiled for 10 min in loading buffer (50 mM Tris–HCl pH6.8, 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol and 1% (v/v) b-mercaptoethanol). The samples were loaded onto a 10% SDS–polyacrylamide gel, electrophoresed at a constant current of 15 mA for 2 h. Gel was stained using 0.1% (w/v) coomassie brilliant blue R250 for 2 h then destained using 45% (v/v) methanol with 10% (v/v) acetic acid for 2 h. The bioactivity of refolded ITGB1-head was assessed under Mn2+ stimulation and shown by native-PAGE. Briefly, refolded protein or BSA (as negative control, BSA Standard, Beyotime Institute of Biotechnology) was treated with 3 mM MnCl2 at 37 °C for 1 h. Treated samples and their controls were mixed with native-PAGE loading buffer (50 mM Tris–HCl pH6.8, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol), then loaded onto the 10% native polyacrylamide gel to separate at 4 °C for 2 h. Specific ITGB1-head bands were shown by Western blotting as described below. The BSA bands were shown using coomassie brilliant blue R250 stain.
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Western blotting
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For Western blotting, after the samples had been separated, the proteins on the gel were transferred onto a polyvinylidene
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difluoride (PVDF) membrane. The membrane was blocked in 5% non-fat dry milk powder, then incubated with mouse anti-his-tag mAb (Proteintech, Chicago, USA) or mouse anti-integrin b1 mAb (clone 18/CD29, BD Biosciences) at 4 °C overnight, followed by incubation with HRP labeled-goat anti-mouse secondary antibody (ZSGB-bio, Beijing, China) at 37 °C for 2 h. Documentation of blots was performed by Canon CanoScan 8800F scanner.
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Circular dichroism
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Circular dichroism was used to determine the secondary structure of the refolded ITGB1-head. The recombinant polypeptide (1 108 M) was pipetted into a 1 mm light path cuvette and scanned on a Bio-logic MOS450 spectropolarimeter from 200 to 300 nm. The scan was repeated 4 times. PBS as the reference sample was subtracted in all cases. Plots were smoothed and exported by SciDAVis ver. 1.D005 (http://scidavis.sourceforge.net/). The contents of a-helix, b-sheet of ITGB1-head were analyzed using K2D3 [16].
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Stability assay
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For thermo-stability assay, equivalent refolded proteins were stored at 80, 20, 4 and 37 °C for 2 d or 1 week, respectively. To test repeated freeze–thaw stability of the recombinant ITGB1head, equivalent proteins were stored at 80 °C constantly or freeze–thawed from 80 °C to room temperature 10 times. Samples were boiled and separated by SDS–PAGE then stained with coomassie brilliant blue and destained as above.
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Results
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Cloning and expression of recombinant polypeptide
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To clone the coding sequence of integrin b1-head piece (ITGB1head), total RNA was extracted from NCM460 normal human colon epithelial cells and cDNA was synthesized. After PCR amplification, a 1356 bp product was successfully obtained (Fig. 1A). The gel-purified PCR product was ligated into a blunt-ended vector and transformed into E. coli cells to allow amplification of the recombinant plasmid (Fig. 1B). This recombinant plasmid and pET-28a vector were then restriction digested to release the ITGB1-head insert and prepare the vector (Fig. 1C) for ligation and transformation. Recombinant plasmid DNA was successfully
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3
Fig. 2. Expression of ITGB1-head. ITGB1-head was induced by 1 mM IPTG at 37 °C for 3 h (lane 3 and 4); lane 1 and 2: the controls with no IPTG added. S: supernatant, P: pellet.
constructed as confirmed by restriction digestion (Fig. 1D) and DNA sequencing (data not shown).
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Expression, purification and refolding of inclusion bodies
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To optimize the soluble expression conditions of integrin b1 head-piece (ITGB1-head), various IPTG concentrations and induction temperatures were assessed. However, the target proteins were mainly expressed in the form of inclusion bodies (Fig. S1), so 1 mM IPTG induction at 37 °C was chosen to obtain more inclusion bodies (Fig. 2). The pelleted bacteria were lysed by sonication, and then the inclusion bodies were concentrated by washing three times with wash buffer (containing 2 M urea). The results showed that irrelevant proteins were reduced largely through the washing process with no significant influence on the recombinant protein (Fig. 3A, lane 1–6). Next, the inclusion bodies were dissolved in extraction buffer (containing 8 M urea, Fig. 3A, lane 7 and 8). The content of recombinant protein was increased from 13% in the initial pellets to 59% in 8 M urea solution (Fig. 3A, lane 2 compared to lane 8, based on the band intensity analysis of the SDS–PAGE). Moreover, the mobility of dissolved ITGB1-head polypeptide was slower than that of the corresponding undissolved one. This observation suggested that the dissolved ITGB1-head was denatured, having a more stretched structure in 8 M urea (ITGB1-head in lane 8 compared to that in other lanes). Ni2+ affinity chromatography was employed here to purify the target polypeptide. Dissolved his-tagged-ITGB1-head was bound to the Ni2+ chelated beads. A series of imidazole concentrations
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Fig. 1. Construction of the recombinant expression plasmid. (A) After PCR amplification, the coding sequence of integrin b1-head piece was successfully obtained. (B) ITGB1head was successfully inserted into blunt-end vector as confirmed by colony PCR; lane 1: PCR products amplified by M13 primers. (C) Both the pET-28a and recombinant blunt-end vectors were digested by Bam HI and Xho I; lane 1: pET-28a vector (before digestion), lane 2: pET-28a vector (after digestion), lane 3: recombinant blunt-end plasmid (after digestion). (D) Restriction digestion of recombinant expression plasmids; Mr: marker.
Please cite this article in press as: T. Shi et al., Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.007
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T. Shi et al. / Protein Expression and Purification xxx (2014) xxx–xxx Table 2 Enrichment of ITGB1-head fusion protein after each step of the purification procedure.
Total cell lysate 1st wash 2nd wash 3rd wash Denaturing buffer Ni–NTA purification Dialysis
Fig. 3. Purification and refolding of ITGB1-head polypeptides. (A) After 3 washes the inclusion bodies were dissolved in extraction buffer. Lane 1 and 2, supernatant and pellet of total cell lysate; lane 3 and 4, supernatant and pellet after the 1st wash; lane 5 and 6, supernatant and pellet after the 2nd wash; lane 7, the supernatant after the 3rd wash; lane 8, the pellet after the 3rd wash (dissolved in extraction buffer); lane 9, undissolved debris in extraction buffer. (B) ITGB1-head proteins were purified by nickel ion-chelated beads under denaturing conditions. The bound proteins were eluted by a concentration gradient of imidazole. (C) The ITGB1-head polypeptides in refolding buffers (containing indicated concentrations of urea) were pipetted from the dialysis bag, centrifuged at 4500g (swing-bucket rotor), then the same volume of supernatant was boiled with loading buffer containing SDS and b-mercaptoethanol and loaded onto a SDS–polyacrylamide gel to separate.
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was used to elute this protein. The results showed that (Fig. 3B) ITGB1-head was mainly eluted by elution buffer containing 50 mM imidazole, a few proteins were found in the 100 mM imidazole elution buffer and no fractions were detected in the buffers containing 150, 300 or 500 mM of imidazole. Based on these results, proteins in 50 mM imidazole elution buffer were collected for the further refolding process. Through stepwise dialyses using refolding buffer containing 4, 2, 1 and 0 M urea, the recombinant ITGB1-head was successfully dissolved in PBS. Fig. 3C showed the status of the recombinant proteins in different refolding buffers. It was obvious that the mobility of the proteins in refolding buffers containing 2 and 1 M urea and pure PBS was a little bit faster than that in other buffers. This suggested that the structure of ITGB1-head was no longer stretched and had been refolded. The purity of the refolded proteins in PBS was more than 98% and the yield was 5.4 mg/L of culture, as determined by SDS–PAGE and BCA method respectively (Table 2). To verify integrity of the refolded ITGB1-head proteins, Western blotting was carried out with anti-his-tag mAb and anti-integrin b1 mAb (clone 18/CD29). The results showed that both of the epitopes
Total protein (mg)
Target protein (mg)
Content of ITGB1-head (%)
12010.00 3152.26 1369.17 703.11 508.74 16.79 5.50
823.15 783.56 570.20 300.11 300.11 15.84 5.40
6.85 24.86 41.65 42.68 58.99 94.36 98.26
were included in the refolded ITGB1-head protein, indicating the integrity of the target proteins (Fig. 4A). Circular dichroism spectrum (CD) is a useful tool to study protein refolding [17], thus we employed CD here to determine the secondary structure of the refolded ITGB1-head. The spectra showed a smooth curve with a negative peak at 205 nm (Fig. 4B). The analysis showed a content of 22.3% a-helices and 21.31% b-sheets in the refolded ITGB1-head, which approximately agreed with the crystal structure of the corresponding polypeptide in the protein data bank (PDB: 3VI3, with the contents of 22% a-helices and 26% b-sheets). The above results confirmed that the refolded ITGB1-head has a native secondary structure.
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Bioactivity assay
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Mn2+ is an agonist of the integrins. The integrins would be altered to a more stretched conformation (activated form) under Mn2+ stimulation [1,2,12,18,19]. According to this, a new method was developed to try to examine the bioactivity of recombinant ITGB1-head. Molar excess of Mn2+ was co-incubated with the refolded ITGB1-head polypeptides at 37 °C for 1 h, then loaded onto the native-polyacrylamide gel to show the conformational switch. The results showed a slower mobility of the treated ITGB1-head sample compared to untreated one (Fig. 5), whereas no difference was found between the Mn2+ treated BSA and its control (Fig. S2). Above results showed the specific conformational switch of refolded ITGB1-head under the stimulation of Mn2+, indicated the ITGB1-head had bioactivity (at least partially) of the extracellular region of native integrin b1.
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Stability assay
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To verify the stability of refolded ITGB1-head polypeptide, equal volumes of the samples were treated with different storage temperatures or repeated freeze–thaw cycles. The results indicated that the recombinant proteins degraded gently with time. There was 61% of residual protein after 2 d storage at 37 °C compared to the control. Even after 1 week storage at 37 °C, 28% of the initial proteins remained (Fig. 6A and B). A repeated freeze–thaw assay also showed the stability of refolded proteins. After 10 cycles of freeze–thaw, there were 59% of the proteins remaining (Fig. 6C and D).
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Discussion
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Integrins are transmembrane proteins and usually exist as heterodimers containing a and b subunits. They act as bridges between cell–cell and cell-extracellular matrix, affecting changes of cell survival, proliferation, adhesion and migration etc [12]. Integrin b1 is the most known subunit in the integrin family due to its tight association with cancer progression [5]. The truncated integrin b1 expressed in this study consisted of plexin/semaphorin/
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Fig. 4. Integrity assay of the refolded ITGB1-head. (A) Refolded ITGB1-head with various loading volumes were separated on SDS–PAGE, stained by coomassie brilliant blue R250 (left panel), and blotted with anti-his-tag and anti-integrin b1 mAbs (right panel). (B) Circular dichroism spectra of the refolded ITGB1-head.
2+
Fig. 5. Bioactivity assay. After 1 h co-incubation with Mn the mobility of refolded ITGB1-head was decreased, indicated the bioactivity of this renatured polypeptide. 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
integrin (PSI), hybrid and b-I domains of integrin b1, which was chosen because its crystal structure had been analyzed [15]. Furthermore it is known that the b-I domain is the core of the integrin b1, in that it connects with various a-subunits and is responsible for ligand binding [1]. The PSI domain participates in the formation of ‘‘open’’ interface with the hybrid domain [20]. The expression of this segment will be more beneficial for further studies. Regarding the current data, researchers have tried to overexpress integrin b1 within different eukaryotic expression systems, such as CHO cells (derived from Chinese hamster) [15], SF9 cells (isolated from Spodoptera frugiperda) [21], but hardly found any prokaryotic expression of it. There are several advantages of prokaryotic expression compared to a eukaryotic one, for example, shorter generation time, ease of handling and established fermentation technology with higher yield [22]. Thus, we tried to express integrin b1 in E. coli cells. The pET expression system has been used to overexpress exogenous proteins for decades. However, despite extensive efforts, our attempts to express soluble ITGB1-head using pET-28a recombinant plasmid were unsuccessful as target proteins were mainly expressed in the form of inclusion bodies. Therefore, the inclusion bodies were collected, purified and refolded. In this present study, after 3 rounds of washes under mild denaturing conditions, the proportions of irrelevant proteins were significantly decreased and the inclusion bodies were
concentrated. This made the purification process easier. There is a 6 his-tag included in the pET-28a vector. One of the advantages of this tag is allowing recombinant proteins to be purified under denaturing conditions. We found that after Ni-affinity chromatography, unrelated proteins further reduced, this was conducive to an increase in the refolding yield and purity ([23] and the papers cited therein). Aggregation is the major problem in refolding denatured recombinant proteins. Lower protein concentration is helpful in reducing the possibility of occurrence [24]. Thus, the purified recombinant proteins were diluted with extraction buffer just before the refolding step to obtain a concentration less than 0.2 mg/mL. During the refolding process, cysteine residues form inter- or intra-disulfide bonds, which may lead to aggregation [25], or form heterogeneous non-native conformations if the disulfide bonds were mismatched. There are 16 cysteine residues in ITGB1-head (data from NCBI protein: 3VI3_B) [15], so GSH/GSSG was added in the refolding buffer to induce oxidative stress in order to facilitate the formation of the correct disulfide bonds [24]. In addition, 5% (v/v) glycerol was added in the refolding buffer as it is reported as a refolding aid to increase the stability of proteins and efficiently elevating the yield [26]. After stepwise dialyses, 5.4 mg renatured ITGB1-head was obtained from 1 L of culture. The productivity was higher than that expressed by eukaryotes. Tartaglia et al. overexpress integrin aVb5 using HEK293 cells and obtained a yield about 2 mg per cell factory (equivalent to forty 15 cm plates) [14], however the molecular weight of ITGB1-head described here was much less than their protein. The main challenge for exogenous protein expression in E. coli is to obtain native and functional protein. The ITGB1-head polypeptide in this present study was a truncated recombinant protein, thus, more careful assessment was required. The integrity of the purified polypeptides was assessed using Western blotting. The
Please cite this article in press as: T. Shi et al., Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.007
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Fig. 6. Stability assay. (A) The same volumes of renatured ITGB1-head protein were stored at different temperatures for 2 d or 1 week. (B) Digitalized histogram of A. (C) The comparison between the samples stored at 80 °C constantly and that which underwent 10 cycles of freeze–thaw. (D) Digitalized histogram of (C). These experiments were carried out 3 times independently.
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results carried out using anti-his-tag and anti-integrin b1 corroborated our design. Moreover, the secondary conformation data as revealed by CD spectra confirmed that the refolded ITGB1-head had the correct structure. Integrin b1 displays its bioactivity through ‘‘conformational switch’’. In the ‘‘closed’’ conformation the ligand binding site of integrin b1 is bent and placed near the surface of the cell membrane. Mn2+ is an agonist of integrin b1. When Mn2+ binds to the adjacent site of ‘‘metal-ion-dependent adhesion site’’ (ADMIDAS) located in b-I domain, latent integrin b1 would be changed to an ‘‘open’’ conformation [1,18,19]. To verify if the refolded proteins had this activity, we developed a new method, which involved co-incubating proteins with a molar excess of Mn2+. The treated proteins showed a decreased mobility in native-polyacrylamide gel electrophoresis, indicating that the proteins had a more extended conformation compared to untreated ones. This confirmed that the recombinant ITGB1-head had at least partial activity of native integrin b1. The recombinant proteins were always at risk of degradation. Therefore, the stability of the refolded protein was examined using different storage temperatures and repeated freeze–thaw cycles. The results showed that the proteins had considerable stability, demonstrating that the renatured ITGB1-head was stable enough to cope with most biochemical experiments. In conclusion, the present study described a method for expression, purification and refolding of truncated integrin b1 in E. coli.
The recombinant proteins were expressed in the form of inclusion bodies. They displayed the proper secondary structure and activities after renaturation. Therefore, ITGB1-head is suitable to use in applications to further characterize integrin b1.
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Conflict of interest
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The authors declare that there are no conflicts of interest.
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Acknowledgments
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We would like to thank Prof. Yuekui Wang at the Institute of Molecular Science, Shanxi University for his valuable suggestions on circular dichroism. This work was supported by the National Q3 Natural Science Foundation of China (No. 31271516, No. 3120 Q4 1072), Shanxi Province Science Foundation for Youths (20120 21028-4) and Oversea Scientists (20111009/20111010), and Research Fund for the Doctoral Program of Higher Education of China (20111401110011).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pep.2014.11.007.
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References
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Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep 5 6
Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli
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Q1
Tonglin Shi a, Lichao Zhang a, Zhuoyu Li a,b,⇑, Ian P. Newton c, Quanbin Zhang d a
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi 030006, China College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China c Cell and Developmental Biology, University of Dundee, Dundee, Scotland DD1 5EH, UK d Central Laboratory of Taiyuan Central Hospital, No. 1, East Sandao Alley, Xinghualing District, Taiyuan, Shanxi 030009, China b
a r t i c l e
i n f o
Article history: Received 22 September 2014 and in revised form 12 November 2014 Available online xxxx Keywords: Prokaryotic expression Integrin b1 pET-28a vector Inclusion body
a b s t r a c t Integrins are a family of transmembrane receptors and among their members, integrin b1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin b1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin b1. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells and after induction by isopropyl-b-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8 M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS–PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin b. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn2+ stimulation. Ó 2014 Published by Elsevier Inc.
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Introduction
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The integrin family consists of 24 members [1]. Their expression in cells, when triggered, relates to various functions of embryonic development, angiogenesis, cell proliferation, adhesion and migration etc [2,3]. Among these, integrin b1 is one of the most studied subunits. Integrin b1 is expressed in a cell- and tissue-specific manner, but almost universally expressed in tumor cells [4]. Its high expression results in the progression of some types of cancers [5]. Moreover, a2b1 [6], a3b1 [7,8], a5b1 [9,10], or a6b1 [11] have been identified as predictors for cancer metastasis. Integrins exist in two distinct conformations, one of which is ‘‘closed’’ (bent) and the other ‘‘open’’. These correspond to the inactive and activated forms of integrin, respectively [12]. How these two conformations switch is still unknown although research suggests that the relationship between the ligands and integrin
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⇑ Corresponding author at: Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006, China. Tel.: +86 351 7018268; fax: +86 351 7018268. E-mail address: [email protected] (Z. Li).
activation [12], or the signal transduction involved in this process [13] are critical. Being able to obtain enough purified integrin b1 protein is a prerequisite to carrying out these types of studies, especially for in vitro experiments. There have been a few studies focusing on the expression of the extracelluar region of integrin b1 [14], and to the best of our knowledge, none of them utilized a prokaryotic expression system. In present study, an efficient process was shown to produce highly purified, soluble and active integrin b1 ectodomain using Escherichia coli. A gene segment that represented 454 amino acids of the N-terminal of integrin b1 was cloned into pET-28a-c (+) (abbreviated as pET-28a) vector and transformed into E. coli BL21 (DE3) cells. The recombinant polypeptides (53 kD) were expressed as inclusion bodies. The inclusion bodies were purified by nickel affinity resin and refolded through stepwise dialysis. The refolded protein was successfully dissolved in phosphate buffered saline (PBS)1, and the yield was about 5.4 mg/L of culture. We proved that Q2
1 Abbreviations used: PBS, phosphate buffered saline; FBS, fetal bovine serum; IPTG, isopropyl-b-D-thiogalactopyranoside; PVDF, polyvinylidene difluoride.
http://dx.doi.org/10.1016/j.pep.2014.11.007 1046-5928/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: T. Shi et al., Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.007
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the renatured polypeptides had native structure and the correct bioactivities.
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Materials and methods
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Cell culture and cDNA synthesis
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NCM460 normal human colon mucosa epithelial cells were cultured in DMEM/F12 (1:1) culture medium (Hyclone) plus 10% fetal bovine serum (FBS, Sangon biotech, Shanghai, China), and grown in humidified air with 5% CO2. Cells were harvested using a cell scraper. Total RNA was isolated using RNAiso Plus (Takara Biotechnology) and purified according to the standard protocol. The cDNA was synthesized using PrimeScript 1st strand cDNA synthesis kit (Takara) according to the user manual.
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Construction of recombinant plasmid
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The PCR primers used for cloning the ectodomain of integrin b1 were designed (Table 1) according to the protein sequence included in PDB: 3VI3 (chain B) [15]. Restriction sites of Bam HI and Xho I were separately introduced to the forward or reverse primers in order to facilitate downstream operation. The specific target DNA segment was amplified on a thermocycler (Mastercycler personal, Eppendorf) using the following program: denaturation at 98 °C for 5 min, followed by 98 °C for 10 s, 15 s at 55 °C, 72 °C for 2 min for 35 cycles. Products underwent a final extension at 72 °C for 10 min. PrimeSTAR HS DNA Polymerase (Takara) was used to ensure high fidelity and accuracy during amplification. The PCR products were resolved using 1% agarose gel and extracted. Purified segments were ligated into pEASY-Blunt Zero Cloning vector (Transgen, Beijing, China) and transformed into Trans5a competent cells (Transgen). Cells were spread on LB plates containing 0.05 mg/mL kanamycin and cultured at 37 °C overnight. Positive clone was selected and grown in 5 mL LB liquid medium containing kanamycin. The recombinant plasmid DNA was extracted (Sangon, Shanghai, China) and digested using Bam HI and Xho I in buffer K (Takara) at 30 °C for 2 h. After gel extraction and purification, inserts were ligated into pET-28a vector (Novagen), that had been digested with the same restriction enzymes as mentioned above using DNA Ligation Kit Ver. 2.1 (Takara) with a ratio of insert: vector = 10:1 (mol/mol) as per the user manual. Recombinant pET-28a was transformed into BL21 (DE3) E. coli cells. Cells were grown overnight at 37 °C on LB plates with kanamycin. Positive colonies were identified by colony PCR, restriction digestion, and verified by DNA sequencing (Sangon, Shanghai, China).
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Expression of recombinant protein
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Positive colony was selected and used to inoculate 1 L of LB medium containing 50 lg/mL kanamycin. Cells were grown in a shaker at 37 °C until OD600 reached 0.9. Following induction with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) being added into the culture medium for 3 h at 37 °C with 200 rpm shaking, cells were harvested by centrifugation at 8000g, 4 °C for 10 min
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Table 1 Primers used for cloning the coding sequence of integrin b1 ectodomain. Underlined characters indicated the restriction sites, italic characters represented the termination signal of transcription. Primer name
Sequences
Restriction enzyme
ITGB1-head-F
50 -CGC GGA TCC CAA ACA GAT GAA AAT AGA TG
Bam HI
ITGB1-head-R
50 -CCG CTC GAG TTA TTC ACA TTC ACA GAT GTA C
Xho I
and washed with cold phosphate buffered saline (PBS, 0.2 g/L KCl, 3.63 g/L Na2HPO4-12H2O, 0.24 g/L KH2PO4, and 8 g/L NaCl, pH = 8.0). The pellets were resuspended in lysis buffer (containing 5 mM b-mercaptoethanol, 1% (w/v) sodium deoxycholate and 1 mM PMSF in PBS, pH = 8.0) then lysed using ultrasound. Intracellular compounds were isolated by 15,000g centrifugation at 4 °C for 15 min. Pellets were washed three times in wash buffer (2 M urea, 1% (v/v) Triton X-100 in PBS, pH = 8.0), then resuspended in extraction buffer (8 M urea, 0.5 M NaCl, 20 mM imidazole, 5 mM b-mercaptoethanol in PBS, pH = 8.0) and allowed to dissolve overnight at 4 °C.
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ITGB1-head purification and refolding
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Extracted inclusion bodies were centrifuged at 4500g (swingbucket rotor, Eppendorf) for 15 min to remove any insoluble debris. The supernatant was transferred to a new falcon tube. Protein concentration was assessed using the BCA method (Beyotime Institute of Biotechnology, Jiangsu, China), and was regulated to be lower than 0.2 mg/mL by extraction buffer to avoid protein aggregation. The protein solution was mixed with binding buffer (same as the above extraction buffer)-equilibrated Ni Sepharose 6 fast flow (GE healthcare) slurry and shaken gently for 1 h at room temperature. After centrifugation at 800g for 5 min, pellets were mixed with binding buffer at a ratio of 1:1 (v/v). The slurry was loaded on a PD-10 column (GE healthcare). Bound fractions were washed and eluted under denaturing conditions according to manufacturer’s instructions. The purified recombinant polypeptide was stepwise dialyzed at 4 °C with the following refolding buffers: (1) refolding buffer A (4 M urea, 0.1 mM glutathione, 0.01 mM glutathione disulfide, 1 mM EDTA, 5% (v/v) glycerol in PBS, pH = 8.0) for 12 h; (2) refolding buffer B (2 M urea, 0.1 mM glutathione, 0.01 mM glutathione disulfide, 1 mM EDTA, 5% (v/v) glycerol, 0.15 M L-arginine in PBS, pH = 8.0) for 12 h; (3) refolding buffer C (1 M urea, 0.1 mM glutathione, 0.01 mM glutathione disulfide, 1 mM EDTA, 5% (v/v) glycerol, in PBS, pH = 8.0) for 12 h; and (4) refolding buffer D (PBS, pH = 8.0) for 12 h, twice. Concentration of refolded ITGB1-head was determined by BCA protein assay kit.
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Purity and bioactivity assay
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The purity of recombinant ITGB1-head polypeptide was determined by SDS–PAGE. Briefly, 7.5 lg of each refolded protein sample was boiled for 10 min in loading buffer (50 mM Tris–HCl pH6.8, 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol and 1% (v/v) b-mercaptoethanol). The samples were loaded onto a 10% SDS–polyacrylamide gel, electrophoresed at a constant current of 15 mA for 2 h. Gel was stained using 0.1% (w/v) coomassie brilliant blue R250 for 2 h then destained using 45% (v/v) methanol with 10% (v/v) acetic acid for 2 h. The bioactivity of refolded ITGB1-head was assessed under Mn2+ stimulation and shown by native-PAGE. Briefly, refolded protein or BSA (as negative control, BSA Standard, Beyotime Institute of Biotechnology) was treated with 3 mM MnCl2 at 37 °C for 1 h. Treated samples and their controls were mixed with native-PAGE loading buffer (50 mM Tris–HCl pH6.8, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol), then loaded onto the 10% native polyacrylamide gel to separate at 4 °C for 2 h. Specific ITGB1-head bands were shown by Western blotting as described below. The BSA bands were shown using coomassie brilliant blue R250 stain.
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Western blotting
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For Western blotting, after the samples had been separated, the proteins on the gel were transferred onto a polyvinylidene
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Please cite this article in press as: T. Shi et al., Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.007
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difluoride (PVDF) membrane. The membrane was blocked in 5% non-fat dry milk powder, then incubated with mouse anti-his-tag mAb (Proteintech, Chicago, USA) or mouse anti-integrin b1 mAb (clone 18/CD29, BD Biosciences) at 4 °C overnight, followed by incubation with HRP labeled-goat anti-mouse secondary antibody (ZSGB-bio, Beijing, China) at 37 °C for 2 h. Documentation of blots was performed by Canon CanoScan 8800F scanner.
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Circular dichroism
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Circular dichroism was used to determine the secondary structure of the refolded ITGB1-head. The recombinant polypeptide (1 108 M) was pipetted into a 1 mm light path cuvette and scanned on a Bio-logic MOS450 spectropolarimeter from 200 to 300 nm. The scan was repeated 4 times. PBS as the reference sample was subtracted in all cases. Plots were smoothed and exported by SciDAVis ver. 1.D005 (http://scidavis.sourceforge.net/). The contents of a-helix, b-sheet of ITGB1-head were analyzed using K2D3 [16].
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Stability assay
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For thermo-stability assay, equivalent refolded proteins were stored at 80, 20, 4 and 37 °C for 2 d or 1 week, respectively. To test repeated freeze–thaw stability of the recombinant ITGB1head, equivalent proteins were stored at 80 °C constantly or freeze–thawed from 80 °C to room temperature 10 times. Samples were boiled and separated by SDS–PAGE then stained with coomassie brilliant blue and destained as above.
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Results
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Cloning and expression of recombinant polypeptide
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To clone the coding sequence of integrin b1-head piece (ITGB1head), total RNA was extracted from NCM460 normal human colon epithelial cells and cDNA was synthesized. After PCR amplification, a 1356 bp product was successfully obtained (Fig. 1A). The gel-purified PCR product was ligated into a blunt-ended vector and transformed into E. coli cells to allow amplification of the recombinant plasmid (Fig. 1B). This recombinant plasmid and pET-28a vector were then restriction digested to release the ITGB1-head insert and prepare the vector (Fig. 1C) for ligation and transformation. Recombinant plasmid DNA was successfully
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3
Fig. 2. Expression of ITGB1-head. ITGB1-head was induced by 1 mM IPTG at 37 °C for 3 h (lane 3 and 4); lane 1 and 2: the controls with no IPTG added. S: supernatant, P: pellet.
constructed as confirmed by restriction digestion (Fig. 1D) and DNA sequencing (data not shown).
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Expression, purification and refolding of inclusion bodies
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To optimize the soluble expression conditions of integrin b1 head-piece (ITGB1-head), various IPTG concentrations and induction temperatures were assessed. However, the target proteins were mainly expressed in the form of inclusion bodies (Fig. S1), so 1 mM IPTG induction at 37 °C was chosen to obtain more inclusion bodies (Fig. 2). The pelleted bacteria were lysed by sonication, and then the inclusion bodies were concentrated by washing three times with wash buffer (containing 2 M urea). The results showed that irrelevant proteins were reduced largely through the washing process with no significant influence on the recombinant protein (Fig. 3A, lane 1–6). Next, the inclusion bodies were dissolved in extraction buffer (containing 8 M urea, Fig. 3A, lane 7 and 8). The content of recombinant protein was increased from 13% in the initial pellets to 59% in 8 M urea solution (Fig. 3A, lane 2 compared to lane 8, based on the band intensity analysis of the SDS–PAGE). Moreover, the mobility of dissolved ITGB1-head polypeptide was slower than that of the corresponding undissolved one. This observation suggested that the dissolved ITGB1-head was denatured, having a more stretched structure in 8 M urea (ITGB1-head in lane 8 compared to that in other lanes). Ni2+ affinity chromatography was employed here to purify the target polypeptide. Dissolved his-tagged-ITGB1-head was bound to the Ni2+ chelated beads. A series of imidazole concentrations
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Fig. 1. Construction of the recombinant expression plasmid. (A) After PCR amplification, the coding sequence of integrin b1-head piece was successfully obtained. (B) ITGB1head was successfully inserted into blunt-end vector as confirmed by colony PCR; lane 1: PCR products amplified by M13 primers. (C) Both the pET-28a and recombinant blunt-end vectors were digested by Bam HI and Xho I; lane 1: pET-28a vector (before digestion), lane 2: pET-28a vector (after digestion), lane 3: recombinant blunt-end plasmid (after digestion). (D) Restriction digestion of recombinant expression plasmids; Mr: marker.
Please cite this article in press as: T. Shi et al., Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.007
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T. Shi et al. / Protein Expression and Purification xxx (2014) xxx–xxx Table 2 Enrichment of ITGB1-head fusion protein after each step of the purification procedure.
Total cell lysate 1st wash 2nd wash 3rd wash Denaturing buffer Ni–NTA purification Dialysis
Fig. 3. Purification and refolding of ITGB1-head polypeptides. (A) After 3 washes the inclusion bodies were dissolved in extraction buffer. Lane 1 and 2, supernatant and pellet of total cell lysate; lane 3 and 4, supernatant and pellet after the 1st wash; lane 5 and 6, supernatant and pellet after the 2nd wash; lane 7, the supernatant after the 3rd wash; lane 8, the pellet after the 3rd wash (dissolved in extraction buffer); lane 9, undissolved debris in extraction buffer. (B) ITGB1-head proteins were purified by nickel ion-chelated beads under denaturing conditions. The bound proteins were eluted by a concentration gradient of imidazole. (C) The ITGB1-head polypeptides in refolding buffers (containing indicated concentrations of urea) were pipetted from the dialysis bag, centrifuged at 4500g (swing-bucket rotor), then the same volume of supernatant was boiled with loading buffer containing SDS and b-mercaptoethanol and loaded onto a SDS–polyacrylamide gel to separate.
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was used to elute this protein. The results showed that (Fig. 3B) ITGB1-head was mainly eluted by elution buffer containing 50 mM imidazole, a few proteins were found in the 100 mM imidazole elution buffer and no fractions were detected in the buffers containing 150, 300 or 500 mM of imidazole. Based on these results, proteins in 50 mM imidazole elution buffer were collected for the further refolding process. Through stepwise dialyses using refolding buffer containing 4, 2, 1 and 0 M urea, the recombinant ITGB1-head was successfully dissolved in PBS. Fig. 3C showed the status of the recombinant proteins in different refolding buffers. It was obvious that the mobility of the proteins in refolding buffers containing 2 and 1 M urea and pure PBS was a little bit faster than that in other buffers. This suggested that the structure of ITGB1-head was no longer stretched and had been refolded. The purity of the refolded proteins in PBS was more than 98% and the yield was 5.4 mg/L of culture, as determined by SDS–PAGE and BCA method respectively (Table 2). To verify integrity of the refolded ITGB1-head proteins, Western blotting was carried out with anti-his-tag mAb and anti-integrin b1 mAb (clone 18/CD29). The results showed that both of the epitopes
Total protein (mg)
Target protein (mg)
Content of ITGB1-head (%)
12010.00 3152.26 1369.17 703.11 508.74 16.79 5.50
823.15 783.56 570.20 300.11 300.11 15.84 5.40
6.85 24.86 41.65 42.68 58.99 94.36 98.26
were included in the refolded ITGB1-head protein, indicating the integrity of the target proteins (Fig. 4A). Circular dichroism spectrum (CD) is a useful tool to study protein refolding [17], thus we employed CD here to determine the secondary structure of the refolded ITGB1-head. The spectra showed a smooth curve with a negative peak at 205 nm (Fig. 4B). The analysis showed a content of 22.3% a-helices and 21.31% b-sheets in the refolded ITGB1-head, which approximately agreed with the crystal structure of the corresponding polypeptide in the protein data bank (PDB: 3VI3, with the contents of 22% a-helices and 26% b-sheets). The above results confirmed that the refolded ITGB1-head has a native secondary structure.
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Bioactivity assay
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Mn2+ is an agonist of the integrins. The integrins would be altered to a more stretched conformation (activated form) under Mn2+ stimulation [1,2,12,18,19]. According to this, a new method was developed to try to examine the bioactivity of recombinant ITGB1-head. Molar excess of Mn2+ was co-incubated with the refolded ITGB1-head polypeptides at 37 °C for 1 h, then loaded onto the native-polyacrylamide gel to show the conformational switch. The results showed a slower mobility of the treated ITGB1-head sample compared to untreated one (Fig. 5), whereas no difference was found between the Mn2+ treated BSA and its control (Fig. S2). Above results showed the specific conformational switch of refolded ITGB1-head under the stimulation of Mn2+, indicated the ITGB1-head had bioactivity (at least partially) of the extracellular region of native integrin b1.
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Stability assay
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To verify the stability of refolded ITGB1-head polypeptide, equal volumes of the samples were treated with different storage temperatures or repeated freeze–thaw cycles. The results indicated that the recombinant proteins degraded gently with time. There was 61% of residual protein after 2 d storage at 37 °C compared to the control. Even after 1 week storage at 37 °C, 28% of the initial proteins remained (Fig. 6A and B). A repeated freeze–thaw assay also showed the stability of refolded proteins. After 10 cycles of freeze–thaw, there were 59% of the proteins remaining (Fig. 6C and D).
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Discussion
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Integrins are transmembrane proteins and usually exist as heterodimers containing a and b subunits. They act as bridges between cell–cell and cell-extracellular matrix, affecting changes of cell survival, proliferation, adhesion and migration etc [12]. Integrin b1 is the most known subunit in the integrin family due to its tight association with cancer progression [5]. The truncated integrin b1 expressed in this study consisted of plexin/semaphorin/
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Fig. 4. Integrity assay of the refolded ITGB1-head. (A) Refolded ITGB1-head with various loading volumes were separated on SDS–PAGE, stained by coomassie brilliant blue R250 (left panel), and blotted with anti-his-tag and anti-integrin b1 mAbs (right panel). (B) Circular dichroism spectra of the refolded ITGB1-head.
2+
Fig. 5. Bioactivity assay. After 1 h co-incubation with Mn the mobility of refolded ITGB1-head was decreased, indicated the bioactivity of this renatured polypeptide. 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
integrin (PSI), hybrid and b-I domains of integrin b1, which was chosen because its crystal structure had been analyzed [15]. Furthermore it is known that the b-I domain is the core of the integrin b1, in that it connects with various a-subunits and is responsible for ligand binding [1]. The PSI domain participates in the formation of ‘‘open’’ interface with the hybrid domain [20]. The expression of this segment will be more beneficial for further studies. Regarding the current data, researchers have tried to overexpress integrin b1 within different eukaryotic expression systems, such as CHO cells (derived from Chinese hamster) [15], SF9 cells (isolated from Spodoptera frugiperda) [21], but hardly found any prokaryotic expression of it. There are several advantages of prokaryotic expression compared to a eukaryotic one, for example, shorter generation time, ease of handling and established fermentation technology with higher yield [22]. Thus, we tried to express integrin b1 in E. coli cells. The pET expression system has been used to overexpress exogenous proteins for decades. However, despite extensive efforts, our attempts to express soluble ITGB1-head using pET-28a recombinant plasmid were unsuccessful as target proteins were mainly expressed in the form of inclusion bodies. Therefore, the inclusion bodies were collected, purified and refolded. In this present study, after 3 rounds of washes under mild denaturing conditions, the proportions of irrelevant proteins were significantly decreased and the inclusion bodies were
concentrated. This made the purification process easier. There is a 6 his-tag included in the pET-28a vector. One of the advantages of this tag is allowing recombinant proteins to be purified under denaturing conditions. We found that after Ni-affinity chromatography, unrelated proteins further reduced, this was conducive to an increase in the refolding yield and purity ([23] and the papers cited therein). Aggregation is the major problem in refolding denatured recombinant proteins. Lower protein concentration is helpful in reducing the possibility of occurrence [24]. Thus, the purified recombinant proteins were diluted with extraction buffer just before the refolding step to obtain a concentration less than 0.2 mg/mL. During the refolding process, cysteine residues form inter- or intra-disulfide bonds, which may lead to aggregation [25], or form heterogeneous non-native conformations if the disulfide bonds were mismatched. There are 16 cysteine residues in ITGB1-head (data from NCBI protein: 3VI3_B) [15], so GSH/GSSG was added in the refolding buffer to induce oxidative stress in order to facilitate the formation of the correct disulfide bonds [24]. In addition, 5% (v/v) glycerol was added in the refolding buffer as it is reported as a refolding aid to increase the stability of proteins and efficiently elevating the yield [26]. After stepwise dialyses, 5.4 mg renatured ITGB1-head was obtained from 1 L of culture. The productivity was higher than that expressed by eukaryotes. Tartaglia et al. overexpress integrin aVb5 using HEK293 cells and obtained a yield about 2 mg per cell factory (equivalent to forty 15 cm plates) [14], however the molecular weight of ITGB1-head described here was much less than their protein. The main challenge for exogenous protein expression in E. coli is to obtain native and functional protein. The ITGB1-head polypeptide in this present study was a truncated recombinant protein, thus, more careful assessment was required. The integrity of the purified polypeptides was assessed using Western blotting. The
Please cite this article in press as: T. Shi et al., Expression, purification and renaturation of truncated human integrin b1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.007
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Fig. 6. Stability assay. (A) The same volumes of renatured ITGB1-head protein were stored at different temperatures for 2 d or 1 week. (B) Digitalized histogram of A. (C) The comparison between the samples stored at 80 °C constantly and that which underwent 10 cycles of freeze–thaw. (D) Digitalized histogram of (C). These experiments were carried out 3 times independently.
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results carried out using anti-his-tag and anti-integrin b1 corroborated our design. Moreover, the secondary conformation data as revealed by CD spectra confirmed that the refolded ITGB1-head had the correct structure. Integrin b1 displays its bioactivity through ‘‘conformational switch’’. In the ‘‘closed’’ conformation the ligand binding site of integrin b1 is bent and placed near the surface of the cell membrane. Mn2+ is an agonist of integrin b1. When Mn2+ binds to the adjacent site of ‘‘metal-ion-dependent adhesion site’’ (ADMIDAS) located in b-I domain, latent integrin b1 would be changed to an ‘‘open’’ conformation [1,18,19]. To verify if the refolded proteins had this activity, we developed a new method, which involved co-incubating proteins with a molar excess of Mn2+. The treated proteins showed a decreased mobility in native-polyacrylamide gel electrophoresis, indicating that the proteins had a more extended conformation compared to untreated ones. This confirmed that the recombinant ITGB1-head had at least partial activity of native integrin b1. The recombinant proteins were always at risk of degradation. Therefore, the stability of the refolded protein was examined using different storage temperatures and repeated freeze–thaw cycles. The results showed that the proteins had considerable stability, demonstrating that the renatured ITGB1-head was stable enough to cope with most biochemical experiments. In conclusion, the present study described a method for expression, purification and refolding of truncated integrin b1 in E. coli.
The recombinant proteins were expressed in the form of inclusion bodies. They displayed the proper secondary structure and activities after renaturation. Therefore, ITGB1-head is suitable to use in applications to further characterize integrin b1.
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Conflict of interest
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The authors declare that there are no conflicts of interest.
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Acknowledgments
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We would like to thank Prof. Yuekui Wang at the Institute of Molecular Science, Shanxi University for his valuable suggestions on circular dichroism. This work was supported by the National Q3 Natural Science Foundation of China (No. 31271516, No. 3120 Q4 1072), Shanxi Province Science Foundation for Youths (20120 21028-4) and Oversea Scientists (20111009/20111010), and Research Fund for the Doctoral Program of Higher Education of China (20111401110011).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pep.2014.11.007.
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