- Email: [email protected]
Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography
Available online at www.sciencedirect.com
Protein Expression and Purification 59 (2008) 138–143 www.elsevier.com/locate/yprep
Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography Ran Zhuang a,1, Yuan Zhang a,1, Rui Zhang b, Chaojun Song a, Kun Yang a, Angang Yang a,b, Boquan Jin a,* b
a Department of Immunology, The Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, PR China Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, PR China
Received 26 November 2007, and in revised form 20 January 2008 Available online 8 February 2008
Abstract GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Affinity purification; GFP; High purity; Monoclonal antibody
The green fluorescent protein (GFP)2, originally isolated from the bioluminescent jellyfish Aequorea victoria, has become one of the most widely studied and exploited proteins in biochemistry and cell biology [1–4]. GFP is a 27 kDa protein, containing 238 amino acid residues, and is able to emit intense and stable fluorescence, without any cofactors, in many different organisms. GFP fluorescence is produced when energy is transferred from the Ca2+-activated photoprotein aequorin to GFP. It is highly stable and resistant to many biological denaturants, including most proteases, pH effects (5–12), temperature *
Corresponding author. Fax: +86 29 83253816. E-mail address: [email protected] (B. Jin). 1 These authors contributed equally to this work. 2 Abbreviations used: GFP, green fluorescent protein; LB, Luria–Bertani; SN, supernatant; SC, subcutaneous. 1046-5928/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.01.020
(Tm = 78 °C), and chaotropic agent (8 M urea) [5]. Given the fact that many species, such as bacterial (Escherichia coli), fungi (Dictyostelium), plant (tobacco), and animal (including mammalian) cells, can express recombinant GFP (rGFP), it has been extensively used in a variety of assays. It is an ideal marker of gene expression, and has been widely used for tracking the localization of target proteins in intact cells, living or fixed tissues, and organisms, or the analysis of molecular interactions, among others [6,7]. With its continued use, several reports on GFP purification methods have emerged, including hydrophobic interaction, size-exclusion and ion-exchange chromatography, phase partitioning, organic solvent extraction, and salt and metal precipitation [8–11]. However, the majority of these methods aim at purification of GFP alone, as opposed to GFP fusion proteins. Foreign proteins, especially large proteins with high molecular weight, can affect
R. Zhuang et al. / Protein Expression and Purification 59 (2008) 138–143
the chemical and physical characteristics of GFP, affecting GFP fusion protein purification. This is critical given the importance of reagent purity in cell growth and biological reactions and exemplified by the fact that many traditional purification methods are limited in their use for biological research given their lower purity. Here, we demonstrate a novel method, based on high specificity and affinity of monoclonal antibody against GFP, for purification of GFP and GFP fusion proteins. The binding of the monoclonal antibody to the GFP epitope allows isolation of GFP, as well as many active GFP-fused recombinant proteins, directly and easily from crude cellular sources, utilizing a mAb-coupled affinity column. Most importantly, the purity of rGFP by this method is superior to other recently described methods. Materials and methods Bacterial strains, vector, reagents and molecular methods Escherichia coli DH5a was used as a host for the propagation of recombinant forms of plasmids pTat, pTat-GFP and for the expression of recombinant GFP protein. E. coli strain was cultivated in Luria–Bertani (LB) broth, with or without 1.5% agar and with the addition of sodium ampicillin (120 lg/ml) when required. The pTat vector was kindly provided by Prof. Zhen RF (Department of Pathogen Biology, the Fourth Military Medical University, Xi’an, PRC). Restriction endonucleases and T4 DNA ligation Kit were purchased from TaKaRa (Dalian, China). DNA was prepared with the Wizard genomic DNA purification Kit (Promega) following manufacturer’s instructions. Plasmid isolation and DNA purification were performed with ¨ CTA FPLC protein purificakits from Biotech, China. A tion system, Q SepharoseTM Fast Flow chromatography column and CNBr-activated Sepharose 4B gel were purchased from GE Healthcare, USA. DNA ligation, restriction endonuclease digestion and gel electrophoresis were performed according to standard techniques or following manufacturer’s instructions. Plasmid was introduced into E. coli strains by chemical transformation. The inserted sequence was confirmed by DNA sequencing. Cloning The GFP gene was amplified using the pEGFP-C3 vector as template and with primers incorporating NdeI and XhoI restriction sites. The amplified DNA was inserted into the pMD18-T vector then sub-cloned into the pTat expression vector containing a Tat encoding sequence, obtaining the vector named pTat-GFP, which was able to constitutively produce GFP-tagged fusion proteins.
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Recombinant protein expression and purification by Q Fast Flow column The pTat-GFP vector was transformed into competent E. coli strain DH5a. Transformed liquid cultures were spread on LB broth with 1.5% agar plates containing 120 lg/ml ampicillin. Single colonies possessing ampicillin resistance were picked and cultured in LB containing 120 lg/ml ampicillin that was subsequently inoculated with 1% (v/v) of the overnight seed culture and cultivated (37 °C, 36 h). GFP protein expression was constitutive, without any induction. Transformed cells were then collected from the growth media by centrifugation (20 min, 8000g). Cell pellets were washed with Washing buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA) and resuspended in Buffer A (50 mM Tris–HCl, pH 8.0; 10 ml per 100 ml of initial culture LB medium, OD400 = 0.8). Cells were lysed (about 1.3 g wet weight cells per 10 ml Buffer A) by snap freeze/ thaw (3) followed by sonication. Following centrifugation (12,000g, 15 min), the lysate supernatant (SN) was loaded onto a Q SepharoseTM Fast Flow column (column ¨ KTA FPLC bed 10 ml) equilibrated in Buffer A using an A System (GE Healthcare, USA), and washed with Buffer A (1 ml/min) until A280 of the flow through reached a stable value. Proteins were eluted by continuous gradient salt concentration with 0–100% Buffer B (50 mM Tris–HCl, 1 M NaCl, pH 8.0) at 1 ml/min), fractions were collected and protein purity was assessed utilizing a 12% SDS– PAGE and Coomassie Brilliant Blue R250 staining. Preparation of mAb against GFP Female BALB/c mice (8-week-old) were immunized with 10 lg of Q SepharoseTM Fast Flow column purified GFP recombinant protein in complete Freund’s adjuvant by subcutaneous (s.c.) injection. Subsequently, immunizations were carried out twice with 10 lg of the GFP protein in incomplete Freud’s adjuvant by s.c. and intraperitoneal (i.p.) injection, respectively, at 3-week intervals. Mice were bled from caudal vein and serum titers determined by ELISA 2 weeks after the third immunization. Mice with favorable titers were boosted with 10 lg GFP protein by i.p. injection. Three days later, splenocytes from the immunized mice and SP2/0 myeloma cells (cultured in RPMI1640 medium, HyClone, USA, containing 20% fetal bovine serum, Gibco/Invitrogen, USA) were fused in the presence of PEG (MW4000, Merck, Germany). Positive hybrids were selected by indirect ELISA and sub-cloned four times using the limiting dilution method. Monoclonal antibodies were produced either from supernatants of the hybridoma culture or from ascites fluid of BALB/c mice in which hybridoma had been i.p. injected. Monoclonal antibodies ¨ CTA Q SepharoseTM were purified from mouse ascites by A Fast Flow chromatography, as described by the manufacturer.
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Indirect ELISA ELISA plates were coated with purified GFP protein (5 lg/ml), washed 3 times with PBST (0.05% Tween-20, 0.13 M NaCl, 0.01 M Na2HPO4, 0.01 M NaH2PO4, pH 7.4) followed by the addition of the hybridoma cell culture supernatant, another round of washing with PBST then detection by HRP-conjugate goat-anti-mouse IgG (DAKO, USA, diluted by 0.5% BSA/PBS). Total DH5a lysate soluble proteins were used in indirect ELISA to eliminate hybridomas secreting mAbs against bacterial components. Characterization of the mAbs against GFP Antibodies were assessed for recognition of GFP recombinant protein by Western blot analysis and immunocytochemical staining. Briefly, a pEGFP-CD226 transfected CHO cell line which expressed the CD226-GFP C-terminal fusion protein on the cell membrane was cultured, in a 6well plate, in RPMI-1640 medium containing 10% fetal bovine serum (data not shown) and monolayers were then fixed (4% paraformaldehyde, 15 min, room temperature), incubated with anti-GFP mAbs and Cy3-conjugated goat-anti-mouse Ig and analyzed by fluorescence microscopy. Immunoprecipitation To detect whether GFP mAbs could bind the soluble recombinant GFP protein, immunoprecipitation was performed as describe previously [12]. Briefly, pTat-GFP transfected DH5a cells were harvested and washed twice with Washing buffer. The supernatant was removed and the pellet resuspend (1 ml of cold Washing buffer per 10 ml of initial culture LB medium, about 1.3 g wet weight cells per 10 ml Washing Buffer). Cells were lysed by snap freeze/thaw (3) followed by sonication. Following centrifugation (12,000g, 15 min), 1 ml of supernatant was gently collected, without disturbing the pellet, and transferred to a clean Eppendorf tube containing Sepharose-Protein G slurry (50 ll of 1:1 slurry, Gibco, USA) and incubated on ice (60 min), centrifuged (10,000g, 2 min, 4 °C) and the supernatant transferred to a fresh Eppendorf tube and incubated (60 min) with anti-GFP mAbs (10 lg, FMU-GFP.3 and FMU-GFP.5). Sepharose-Protein G slurry (50 ll) was then added to the lysate and incubated (4 °C, 2 h, on a rotator), centrifuged (10,000g, 30 s, 4 °C) and the supernatant carefully removed and the beads washed 3–5 times with Lysis buffer (500 ll). The supernatant was then aspirated and 40 ll of 2 Laemmli sample buffer was added to the bead pellet, vortexed, heated (90–100 °C, 10 min) and loaded onto a 12% SDS–PAGE gel that was then stained with Coomassie Brilliant Blue R250 for visual analysis of the immunoprecipitated protein.
Purification of GFP fusion protein by affinity column chromatography The mAb with higher affinity, as evaluated by ELISA and immunoprecipitation assays, was coupled to the CNBr-activated Sepharose 4B resin for preparation of the affinity column, following manufacturer’s instruction. Briefly, the mAb was purified by QFF chromatography ¨ KTA, GE Healthcare, USA) and dialyzed to column (A coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3). Mix the resin and mAb in a stoppered vessel, and then rotate the mixture end-over-end for 2 h at room temperature. After washed away excess mAb and blocked the remaining active groups by blocking buffer (0.1 M Tris– HCl buffer, pH 8.0), the resin was packed to a column, equilibrated and ready for use. The 5 ml of pTat-GFP transfected DH5a lysate supernatant was then loaded onto the 2 ml GFP mAb-coupled affinity chromatography column, that was then washed with Binding buffer (10 column volumes, PBS, 0.13 M NaCl, 0.01 M Na2HPO4, 0.01 M NaH2PO4, pH 7.4) and eluted with elution buffer (0.1 M glycine–HCl, pH 4.5). Elution fractions were neutralized immediately by addition of a small amount of neutralizing buffer (1 M Tris–HCl, pH 9.0), and dialyzed (0.15 M PBS, pH 7.4). The eluted rGFP samples were analyzed by a discontinuous 12% SDS–PAGE and Coomassie Brilliant Blue R250 stain. Purification and yields were estimated by the determination of protein content using bio-software Bandscan5.0 (www.bio-soft.net). Detection of biological properties of purified GFP in live cells Recombinant GFP was expressed as a fusion protein containing a Tat peptide transduction sequence on the Nterminus, which can lead to non-specific entrance of GFP fusion proteins into cells [13]. Following overnight culture (37 °C, 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum), CHO cells were treated with recombinant Tat-GFP (30 lg/ml) and observed by fluorescence microscopy. At the same time, CHO cells were harvested using 0.02% EDTA/PBS and suspended in RPMI-1640 medium containing 10% fetal bovine serum, followed by treatment with recombinant GFP (30 min, 10, 30, 90 or 180 lg/ml). Fluorescence intensities of treated CHO cells were detected by Flow Cytometric Assay. Results The expression and crude purification of recombinant TatGFP fusion protein The Tat-GFP protein was expressed by E. coli strain DH5a and bacteria lysate was loaded on a Q Fast Flow column (see Methods). The chromatography profile showed a wide peak of material with Buffer A, followed by several peaks eluted by gradient of Buffer B. SDS–
R. Zhuang et al. / Protein Expression and Purification 59 (2008) 138–143
PAGE analysis of the peaks showed that Tat-GFP protein was eluted by Buffer B as the first single sharp peak (Fig. 1). The reactivity of mAbs to the recombinant GFP Two hybridoma cell lines secreting mAbs against GFP were obtained following standard protocols (Table 1). Monoclonal antibodies to Tat-GFP were assessed by Western blot analysis for recognition of denatured rGFP fusion protein. Both clones (FMU-GFP.3 and FMU-GFP.5) showed positive reaction to recombinant GFP protein (Fig. 2). FMU-GFP.3 and FMU-GFP.5 were also used in immunocytochemical staining of pEGFP-CD226 vector transfected CHO cells expressing membrane human CD226 with a C-terminal GFP tag (Fig. 3). Additionally, FMU-GFP.3 and FMU-GFP.5 immunoprecipitated the recombinant soluble GFP protein (Fig. 4). Purification of GFP by immunoaffinity column The FMU-GFP.5 mAb was coupled to CNBr-activated Sepharose 4B to prepare the affinity column. The DH5a lysate supernatant containing Tat-GFP fusion protein was loaded onto the anti-GFP mAb affinity chromatography column, and the rGFP eluted. The SDS–PAGE, Coomassie Brilliant Blue R250 staining (Fig. 5) and biosoftware Bandscan5.0 were used to calculate the purity of the GFP fusion protein, which reached to more than 97%. And the concentration of cell lysate and purified rGFP were determined by ultraviolet spectrophotometer. After one step of affinity purification, including the cell
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Table 1 Characterization of monoclonal antibodies against GFP Clone #
ELISA
WB
IP
IHC
Affinity column
FMU-GFP.3 (IgG2a, j) FMU-GFP.5 (IgG2b, j)
+ +
+ +
+ +
+ +
Did not Work +
Fig. 2. Western blot analysis of the monoclonal antibodies against GFP. The mAbs FMU-GFP.3 and FMU-GFP.5 displayed positive reaction to recombinant GFP at the concentration of 2 lg/ml. No reactivity was observed with normal mouse Ig. Numbers on left = molecular weight markers.
lysate loading onto the column and eluting by elution buffer (0.1 M glycine–HCl, pH 4.5), about 90% of rGFP can be recovered. In addition, to research the signal transduction of CD226, we prepared the Tat-CD226ICD-GFP (intracellular domain of CD226) fusion protein which was also purified by mAb FMU-GFP.5 coupled affinity column successfully. These results were analyzed by SDS–PAGE. The biological characterization of affinity purified Tat-GFP Biological characterization of Tat-GFP recombinant protein utilized rGFP purified by QFF column and affinity column. Green fluorescence of CHO cells demonstrated that Tat-GFP was able to efficiently enter CHO cells in a non-specific manner, no matter the type of purification method used [13]. FACS analysis of protein transduction in CHO cells treated with 10, 30, 90 or 180 lg/ml Tat-GFP recombinant protein revealed a concentration dependence (Fig. 6). Moreover, our data suggested that Tat-GFP entered a variety of cells, such as HEK293 human fibroblasts, K562 lymphoblasts, THP-1 monocytes, BT325 glioma cells, HepG2 hepatocellular carcinoma cells, NIH3T3 mouse fibroblasts, sp2/0 myeloma cells, as well as all human blood cells including T- and B-cells and monocytes (data not shown). Discussion
Fig. 1. Purification of Tat-GFP by Q Sepharose Fast Flow chromatography. Bacterial lysate (200 ml) was loaded onto a QFF chromatography column. A step gradient of 0–100% Buffer B (250 ml) was applied (dashed line) and fractions were collected. A280 (thick line) was also continuously monitored. The recombinant Tat-GFP fusion protein was eluted as the first sharp single peak. Most proteins of the bacterial lysate did not bind to the resin.
Green fluorescent protein, as a fusion partner, has been widely used as an excellent reporter molecule to track the expression or localization of recombinant proteins. Moreover, GFP expressed in E. coli cells also provides a high expression level of many heterologous polypeptides [10]. Many protocols and methods have been developed to purify recombinant GFP fusion proteins [7]. Here, we demonstrated a mAbs-based purification method for GFP fusion proteins. Recombinant GFP was utilized as an
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R. Zhuang et al. / Protein Expression and Purification 59 (2008) 138–143
Fig. 3. Immunohistochemical analysis of anti-GFP mAbs. The mAb FMU-GFP.5 showed strong positive reaction with pEGFP-CD226 vector transfected CHO cells. (A) pEGFP-CD226 vector transfected CHO cells showed green fluorescence on the cell membrane; (B) mAb FMU-GFP.5 and PE conjugated goat-anti-mouse poAb (polyclonal antibody) staining; (C) dual-stain image (A and B). (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
Fig. 5. SDS–PAGE analysis of purification of recombinant GFP fusion proteins by ion-exchange chromatography and mAb affinity column chromatography. Numbers on the left = molecular weight markers.
Fig. 4. Immunoprecipitation analysis of mAb against GFP. The mAbs FMU-GFP.3 and FMU-GFP.5 immunoprecipitated rGFP from E. coli lysate. Numbers on the left = molecular weight markers.
immunogen, and hybridoma two clones, secreting monoclonal antibodies, were raised and characterized. Using mAb FMU-GFP.5-coupled Sepharose 4B resin, we prepared an affinity chromatography column utilizing a simple two-step protocol, effectively purifying both GFP and GFP fusion proteins with a high purity of 97% and a sample yield about 90%, with full biological function (Fig. 6 and Fig. 7). The technique was ideal for capture or as an intermediate step in a purification protocol and can be used with a variety of suitable ligands. Our data suggested that the novel method proposed in this study will be a valuable
Fig. 6. FACS analysis of GFP protein transduction in CHO cell treated with affinity chromatography-purified tat-GFP. FACS analysis of CHO cells treated with Tat-GFP recombinant protein (0, 10, and 30 lg/ml, 1 h) demonstrated the Tat-GFP protein transduction efficiency was concentration dependent.
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References
Fig. 7. Observation of GFP protein transduction in CHO cells treated with affinity chromatography-purified tat-GFP. CHO cells were treated with Tat-GFP recombinant protein (90 lg/ml, 30 min) demonstrated the Tat-GFP protein can enter cells efficiently.
tool for the routine purification of other GFP fusion proteins. Immunoaffinity chromatography has also been utilized in the purification of recombinant human TPB protein, with demonstrated activity observed by gel-shift and transcription assays [14]. Affinity chromatography is a powerful method to purify recombinant proteins from many genera. Recent research by Burgess and Thompson proposed an ingenious method for developing mAbs that were able to bind tightly, but release under gentle elution conditions (polyol-responsive mAb, PR-mAb), ideal properties for use in immunoaffinity chromatography [15]. In response to the rapidly growing field of proteomics, the use of recombinant proteins has also greatly increased in recent years [16]. Recombinant hybrids containing an affinity tag, to facilitate the purification of the target polypeptides or proteins are widely used. In this case, many different proteins, domains, or peptides can be fused with the GFP tag, not only as a monitoring probe, but also as an affinity tag. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (No. 30672370) and the National Key Technologies R&D Program of China.
[1] J. Wiedenmann, G.U. Nienhaus, Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family, Expert Review of Proteomics 3 (2006) 361–374. [2] O. Bjo¨rnberg, H. Ostergaard, J.R. Winther, Measuring intracellular redox conditions using GFP-based sensors, Antioxid Redox Signal 8 (2006) 354–361. [3] C.L. Takanishi, E.A. Bykova, W. Cheng, J. Zheng, GFP-based FRET analysis in live cells, Brain Research 1091 (2006) 132– 139. [4] T.J. Magliery, L. Regan, Reassembled GFP: detecting protein-protein interactions and protein expression patterns, Methods of Biochemical Analysis 47 (2006) 391–405. [5] M. Chalfie, S. Kain, Green Fluorescent Protein: Properties Applications, and Protocols, Wiley Liss, New York, 1998. [6] M.E. Meima, K.E. Weening, P. Schaap, Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium, Protein Expression and Purification 53 (2007) 283–288. [7] G.D. Peckham, R.C. Bugos, W.W. Su, Purification of GFP fusion proteins from transgenic plant cell cultures, Protein Expression and Purification 49 (2006) 183–189. [8] C. Cabanne, A.M. Noubhani, W. Dieryck, A. Hocquellet, X. Santarelli, Evaluation of three expanded bed adsorption anion exchange matrices with the aid of recombinant enhanced green fluorescent protein overexpressed in Escherichia coli, Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences 808 (2004) 91–97. [9] T.C.V. Penna, M. Ishii, A.P. Junior, L. de Oliveira Nascimento, L.C. de Souza, O. Cholewa, Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins, Applied Microbiology and Biotechnology 114 (2004) 453–468. [10] V.S. Skosyrev, N.V. Rudenko, A.V. Yakhnin, V.E. Zagranichny, L.I. Popova, M.V. Zakharov, A.Yu. Gorokhovatsky, Leonid M. Vinokurov, EGFP as a fusion partner for the expression and organic extraction of small polypeptides, Protein Expression and Purification 27 (2003) 55–62. [11] S.R. McRae, C.L. Brown, G.R. Bushell, Rapid purification of EGFP, EYFP, and ECFP with high yield and purity, Protein Expression and Purification 41 (2005) 121–127. [12] E. Harlow, D. Lane (Eds.), Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1999. [13] H. Nagahara, A.M. Vocero-Akbani, E.L. Snyder, A. Ho, D.G. Latham, N.A. Lissy, M. Becker-Hapak, S.A. Ezhevsky, S.F. Dowdy, Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration, Nature Medicine 4 (1998) 1449–1452. [14] N.E. Thompson, K.M. Foley, R.R. Burgess, Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein (TBP) and use in immunoaffinity chromatography, Protein Expression and Purification 36 (2004) 186–197. [15] R.R. Burgess, N.E. Thompson, Advances in gentle immunoaffinity chromatography, Current opinion in Biotechnology 13 (2002) 304– 308. [16] K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Applied Microbiology and Biotechnology 60 (2003) 523–533.
Protein Expression and Purification 59 (2008) 138–143 www.elsevier.com/locate/yprep
Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography Ran Zhuang a,1, Yuan Zhang a,1, Rui Zhang b, Chaojun Song a, Kun Yang a, Angang Yang a,b, Boquan Jin a,* b
a Department of Immunology, The Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, PR China Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, PR China
Received 26 November 2007, and in revised form 20 January 2008 Available online 8 February 2008
Abstract GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Affinity purification; GFP; High purity; Monoclonal antibody
The green fluorescent protein (GFP)2, originally isolated from the bioluminescent jellyfish Aequorea victoria, has become one of the most widely studied and exploited proteins in biochemistry and cell biology [1–4]. GFP is a 27 kDa protein, containing 238 amino acid residues, and is able to emit intense and stable fluorescence, without any cofactors, in many different organisms. GFP fluorescence is produced when energy is transferred from the Ca2+-activated photoprotein aequorin to GFP. It is highly stable and resistant to many biological denaturants, including most proteases, pH effects (5–12), temperature *
Corresponding author. Fax: +86 29 83253816. E-mail address: [email protected] (B. Jin). 1 These authors contributed equally to this work. 2 Abbreviations used: GFP, green fluorescent protein; LB, Luria–Bertani; SN, supernatant; SC, subcutaneous. 1046-5928/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.01.020
(Tm = 78 °C), and chaotropic agent (8 M urea) [5]. Given the fact that many species, such as bacterial (Escherichia coli), fungi (Dictyostelium), plant (tobacco), and animal (including mammalian) cells, can express recombinant GFP (rGFP), it has been extensively used in a variety of assays. It is an ideal marker of gene expression, and has been widely used for tracking the localization of target proteins in intact cells, living or fixed tissues, and organisms, or the analysis of molecular interactions, among others [6,7]. With its continued use, several reports on GFP purification methods have emerged, including hydrophobic interaction, size-exclusion and ion-exchange chromatography, phase partitioning, organic solvent extraction, and salt and metal precipitation [8–11]. However, the majority of these methods aim at purification of GFP alone, as opposed to GFP fusion proteins. Foreign proteins, especially large proteins with high molecular weight, can affect
R. Zhuang et al. / Protein Expression and Purification 59 (2008) 138–143
the chemical and physical characteristics of GFP, affecting GFP fusion protein purification. This is critical given the importance of reagent purity in cell growth and biological reactions and exemplified by the fact that many traditional purification methods are limited in their use for biological research given their lower purity. Here, we demonstrate a novel method, based on high specificity and affinity of monoclonal antibody against GFP, for purification of GFP and GFP fusion proteins. The binding of the monoclonal antibody to the GFP epitope allows isolation of GFP, as well as many active GFP-fused recombinant proteins, directly and easily from crude cellular sources, utilizing a mAb-coupled affinity column. Most importantly, the purity of rGFP by this method is superior to other recently described methods. Materials and methods Bacterial strains, vector, reagents and molecular methods Escherichia coli DH5a was used as a host for the propagation of recombinant forms of plasmids pTat, pTat-GFP and for the expression of recombinant GFP protein. E. coli strain was cultivated in Luria–Bertani (LB) broth, with or without 1.5% agar and with the addition of sodium ampicillin (120 lg/ml) when required. The pTat vector was kindly provided by Prof. Zhen RF (Department of Pathogen Biology, the Fourth Military Medical University, Xi’an, PRC). Restriction endonucleases and T4 DNA ligation Kit were purchased from TaKaRa (Dalian, China). DNA was prepared with the Wizard genomic DNA purification Kit (Promega) following manufacturer’s instructions. Plasmid isolation and DNA purification were performed with ¨ CTA FPLC protein purificakits from Biotech, China. A tion system, Q SepharoseTM Fast Flow chromatography column and CNBr-activated Sepharose 4B gel were purchased from GE Healthcare, USA. DNA ligation, restriction endonuclease digestion and gel electrophoresis were performed according to standard techniques or following manufacturer’s instructions. Plasmid was introduced into E. coli strains by chemical transformation. The inserted sequence was confirmed by DNA sequencing. Cloning The GFP gene was amplified using the pEGFP-C3 vector as template and with primers incorporating NdeI and XhoI restriction sites. The amplified DNA was inserted into the pMD18-T vector then sub-cloned into the pTat expression vector containing a Tat encoding sequence, obtaining the vector named pTat-GFP, which was able to constitutively produce GFP-tagged fusion proteins.
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Recombinant protein expression and purification by Q Fast Flow column The pTat-GFP vector was transformed into competent E. coli strain DH5a. Transformed liquid cultures were spread on LB broth with 1.5% agar plates containing 120 lg/ml ampicillin. Single colonies possessing ampicillin resistance were picked and cultured in LB containing 120 lg/ml ampicillin that was subsequently inoculated with 1% (v/v) of the overnight seed culture and cultivated (37 °C, 36 h). GFP protein expression was constitutive, without any induction. Transformed cells were then collected from the growth media by centrifugation (20 min, 8000g). Cell pellets were washed with Washing buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA) and resuspended in Buffer A (50 mM Tris–HCl, pH 8.0; 10 ml per 100 ml of initial culture LB medium, OD400 = 0.8). Cells were lysed (about 1.3 g wet weight cells per 10 ml Buffer A) by snap freeze/ thaw (3) followed by sonication. Following centrifugation (12,000g, 15 min), the lysate supernatant (SN) was loaded onto a Q SepharoseTM Fast Flow column (column ¨ KTA FPLC bed 10 ml) equilibrated in Buffer A using an A System (GE Healthcare, USA), and washed with Buffer A (1 ml/min) until A280 of the flow through reached a stable value. Proteins were eluted by continuous gradient salt concentration with 0–100% Buffer B (50 mM Tris–HCl, 1 M NaCl, pH 8.0) at 1 ml/min), fractions were collected and protein purity was assessed utilizing a 12% SDS– PAGE and Coomassie Brilliant Blue R250 staining. Preparation of mAb against GFP Female BALB/c mice (8-week-old) were immunized with 10 lg of Q SepharoseTM Fast Flow column purified GFP recombinant protein in complete Freund’s adjuvant by subcutaneous (s.c.) injection. Subsequently, immunizations were carried out twice with 10 lg of the GFP protein in incomplete Freud’s adjuvant by s.c. and intraperitoneal (i.p.) injection, respectively, at 3-week intervals. Mice were bled from caudal vein and serum titers determined by ELISA 2 weeks after the third immunization. Mice with favorable titers were boosted with 10 lg GFP protein by i.p. injection. Three days later, splenocytes from the immunized mice and SP2/0 myeloma cells (cultured in RPMI1640 medium, HyClone, USA, containing 20% fetal bovine serum, Gibco/Invitrogen, USA) were fused in the presence of PEG (MW4000, Merck, Germany). Positive hybrids were selected by indirect ELISA and sub-cloned four times using the limiting dilution method. Monoclonal antibodies were produced either from supernatants of the hybridoma culture or from ascites fluid of BALB/c mice in which hybridoma had been i.p. injected. Monoclonal antibodies ¨ CTA Q SepharoseTM were purified from mouse ascites by A Fast Flow chromatography, as described by the manufacturer.
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Indirect ELISA ELISA plates were coated with purified GFP protein (5 lg/ml), washed 3 times with PBST (0.05% Tween-20, 0.13 M NaCl, 0.01 M Na2HPO4, 0.01 M NaH2PO4, pH 7.4) followed by the addition of the hybridoma cell culture supernatant, another round of washing with PBST then detection by HRP-conjugate goat-anti-mouse IgG (DAKO, USA, diluted by 0.5% BSA/PBS). Total DH5a lysate soluble proteins were used in indirect ELISA to eliminate hybridomas secreting mAbs against bacterial components. Characterization of the mAbs against GFP Antibodies were assessed for recognition of GFP recombinant protein by Western blot analysis and immunocytochemical staining. Briefly, a pEGFP-CD226 transfected CHO cell line which expressed the CD226-GFP C-terminal fusion protein on the cell membrane was cultured, in a 6well plate, in RPMI-1640 medium containing 10% fetal bovine serum (data not shown) and monolayers were then fixed (4% paraformaldehyde, 15 min, room temperature), incubated with anti-GFP mAbs and Cy3-conjugated goat-anti-mouse Ig and analyzed by fluorescence microscopy. Immunoprecipitation To detect whether GFP mAbs could bind the soluble recombinant GFP protein, immunoprecipitation was performed as describe previously [12]. Briefly, pTat-GFP transfected DH5a cells were harvested and washed twice with Washing buffer. The supernatant was removed and the pellet resuspend (1 ml of cold Washing buffer per 10 ml of initial culture LB medium, about 1.3 g wet weight cells per 10 ml Washing Buffer). Cells were lysed by snap freeze/thaw (3) followed by sonication. Following centrifugation (12,000g, 15 min), 1 ml of supernatant was gently collected, without disturbing the pellet, and transferred to a clean Eppendorf tube containing Sepharose-Protein G slurry (50 ll of 1:1 slurry, Gibco, USA) and incubated on ice (60 min), centrifuged (10,000g, 2 min, 4 °C) and the supernatant transferred to a fresh Eppendorf tube and incubated (60 min) with anti-GFP mAbs (10 lg, FMU-GFP.3 and FMU-GFP.5). Sepharose-Protein G slurry (50 ll) was then added to the lysate and incubated (4 °C, 2 h, on a rotator), centrifuged (10,000g, 30 s, 4 °C) and the supernatant carefully removed and the beads washed 3–5 times with Lysis buffer (500 ll). The supernatant was then aspirated and 40 ll of 2 Laemmli sample buffer was added to the bead pellet, vortexed, heated (90–100 °C, 10 min) and loaded onto a 12% SDS–PAGE gel that was then stained with Coomassie Brilliant Blue R250 for visual analysis of the immunoprecipitated protein.
Purification of GFP fusion protein by affinity column chromatography The mAb with higher affinity, as evaluated by ELISA and immunoprecipitation assays, was coupled to the CNBr-activated Sepharose 4B resin for preparation of the affinity column, following manufacturer’s instruction. Briefly, the mAb was purified by QFF chromatography ¨ KTA, GE Healthcare, USA) and dialyzed to column (A coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3). Mix the resin and mAb in a stoppered vessel, and then rotate the mixture end-over-end for 2 h at room temperature. After washed away excess mAb and blocked the remaining active groups by blocking buffer (0.1 M Tris– HCl buffer, pH 8.0), the resin was packed to a column, equilibrated and ready for use. The 5 ml of pTat-GFP transfected DH5a lysate supernatant was then loaded onto the 2 ml GFP mAb-coupled affinity chromatography column, that was then washed with Binding buffer (10 column volumes, PBS, 0.13 M NaCl, 0.01 M Na2HPO4, 0.01 M NaH2PO4, pH 7.4) and eluted with elution buffer (0.1 M glycine–HCl, pH 4.5). Elution fractions were neutralized immediately by addition of a small amount of neutralizing buffer (1 M Tris–HCl, pH 9.0), and dialyzed (0.15 M PBS, pH 7.4). The eluted rGFP samples were analyzed by a discontinuous 12% SDS–PAGE and Coomassie Brilliant Blue R250 stain. Purification and yields were estimated by the determination of protein content using bio-software Bandscan5.0 (www.bio-soft.net). Detection of biological properties of purified GFP in live cells Recombinant GFP was expressed as a fusion protein containing a Tat peptide transduction sequence on the Nterminus, which can lead to non-specific entrance of GFP fusion proteins into cells [13]. Following overnight culture (37 °C, 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum), CHO cells were treated with recombinant Tat-GFP (30 lg/ml) and observed by fluorescence microscopy. At the same time, CHO cells were harvested using 0.02% EDTA/PBS and suspended in RPMI-1640 medium containing 10% fetal bovine serum, followed by treatment with recombinant GFP (30 min, 10, 30, 90 or 180 lg/ml). Fluorescence intensities of treated CHO cells were detected by Flow Cytometric Assay. Results The expression and crude purification of recombinant TatGFP fusion protein The Tat-GFP protein was expressed by E. coli strain DH5a and bacteria lysate was loaded on a Q Fast Flow column (see Methods). The chromatography profile showed a wide peak of material with Buffer A, followed by several peaks eluted by gradient of Buffer B. SDS–
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PAGE analysis of the peaks showed that Tat-GFP protein was eluted by Buffer B as the first single sharp peak (Fig. 1). The reactivity of mAbs to the recombinant GFP Two hybridoma cell lines secreting mAbs against GFP were obtained following standard protocols (Table 1). Monoclonal antibodies to Tat-GFP were assessed by Western blot analysis for recognition of denatured rGFP fusion protein. Both clones (FMU-GFP.3 and FMU-GFP.5) showed positive reaction to recombinant GFP protein (Fig. 2). FMU-GFP.3 and FMU-GFP.5 were also used in immunocytochemical staining of pEGFP-CD226 vector transfected CHO cells expressing membrane human CD226 with a C-terminal GFP tag (Fig. 3). Additionally, FMU-GFP.3 and FMU-GFP.5 immunoprecipitated the recombinant soluble GFP protein (Fig. 4). Purification of GFP by immunoaffinity column The FMU-GFP.5 mAb was coupled to CNBr-activated Sepharose 4B to prepare the affinity column. The DH5a lysate supernatant containing Tat-GFP fusion protein was loaded onto the anti-GFP mAb affinity chromatography column, and the rGFP eluted. The SDS–PAGE, Coomassie Brilliant Blue R250 staining (Fig. 5) and biosoftware Bandscan5.0 were used to calculate the purity of the GFP fusion protein, which reached to more than 97%. And the concentration of cell lysate and purified rGFP were determined by ultraviolet spectrophotometer. After one step of affinity purification, including the cell
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Table 1 Characterization of monoclonal antibodies against GFP Clone #
ELISA
WB
IP
IHC
Affinity column
FMU-GFP.3 (IgG2a, j) FMU-GFP.5 (IgG2b, j)
+ +
+ +
+ +
+ +
Did not Work +
Fig. 2. Western blot analysis of the monoclonal antibodies against GFP. The mAbs FMU-GFP.3 and FMU-GFP.5 displayed positive reaction to recombinant GFP at the concentration of 2 lg/ml. No reactivity was observed with normal mouse Ig. Numbers on left = molecular weight markers.
lysate loading onto the column and eluting by elution buffer (0.1 M glycine–HCl, pH 4.5), about 90% of rGFP can be recovered. In addition, to research the signal transduction of CD226, we prepared the Tat-CD226ICD-GFP (intracellular domain of CD226) fusion protein which was also purified by mAb FMU-GFP.5 coupled affinity column successfully. These results were analyzed by SDS–PAGE. The biological characterization of affinity purified Tat-GFP Biological characterization of Tat-GFP recombinant protein utilized rGFP purified by QFF column and affinity column. Green fluorescence of CHO cells demonstrated that Tat-GFP was able to efficiently enter CHO cells in a non-specific manner, no matter the type of purification method used [13]. FACS analysis of protein transduction in CHO cells treated with 10, 30, 90 or 180 lg/ml Tat-GFP recombinant protein revealed a concentration dependence (Fig. 6). Moreover, our data suggested that Tat-GFP entered a variety of cells, such as HEK293 human fibroblasts, K562 lymphoblasts, THP-1 monocytes, BT325 glioma cells, HepG2 hepatocellular carcinoma cells, NIH3T3 mouse fibroblasts, sp2/0 myeloma cells, as well as all human blood cells including T- and B-cells and monocytes (data not shown). Discussion
Fig. 1. Purification of Tat-GFP by Q Sepharose Fast Flow chromatography. Bacterial lysate (200 ml) was loaded onto a QFF chromatography column. A step gradient of 0–100% Buffer B (250 ml) was applied (dashed line) and fractions were collected. A280 (thick line) was also continuously monitored. The recombinant Tat-GFP fusion protein was eluted as the first sharp single peak. Most proteins of the bacterial lysate did not bind to the resin.
Green fluorescent protein, as a fusion partner, has been widely used as an excellent reporter molecule to track the expression or localization of recombinant proteins. Moreover, GFP expressed in E. coli cells also provides a high expression level of many heterologous polypeptides [10]. Many protocols and methods have been developed to purify recombinant GFP fusion proteins [7]. Here, we demonstrated a mAbs-based purification method for GFP fusion proteins. Recombinant GFP was utilized as an
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Fig. 3. Immunohistochemical analysis of anti-GFP mAbs. The mAb FMU-GFP.5 showed strong positive reaction with pEGFP-CD226 vector transfected CHO cells. (A) pEGFP-CD226 vector transfected CHO cells showed green fluorescence on the cell membrane; (B) mAb FMU-GFP.5 and PE conjugated goat-anti-mouse poAb (polyclonal antibody) staining; (C) dual-stain image (A and B). (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
Fig. 5. SDS–PAGE analysis of purification of recombinant GFP fusion proteins by ion-exchange chromatography and mAb affinity column chromatography. Numbers on the left = molecular weight markers.
Fig. 4. Immunoprecipitation analysis of mAb against GFP. The mAbs FMU-GFP.3 and FMU-GFP.5 immunoprecipitated rGFP from E. coli lysate. Numbers on the left = molecular weight markers.
immunogen, and hybridoma two clones, secreting monoclonal antibodies, were raised and characterized. Using mAb FMU-GFP.5-coupled Sepharose 4B resin, we prepared an affinity chromatography column utilizing a simple two-step protocol, effectively purifying both GFP and GFP fusion proteins with a high purity of 97% and a sample yield about 90%, with full biological function (Fig. 6 and Fig. 7). The technique was ideal for capture or as an intermediate step in a purification protocol and can be used with a variety of suitable ligands. Our data suggested that the novel method proposed in this study will be a valuable
Fig. 6. FACS analysis of GFP protein transduction in CHO cell treated with affinity chromatography-purified tat-GFP. FACS analysis of CHO cells treated with Tat-GFP recombinant protein (0, 10, and 30 lg/ml, 1 h) demonstrated the Tat-GFP protein transduction efficiency was concentration dependent.
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References
Fig. 7. Observation of GFP protein transduction in CHO cells treated with affinity chromatography-purified tat-GFP. CHO cells were treated with Tat-GFP recombinant protein (90 lg/ml, 30 min) demonstrated the Tat-GFP protein can enter cells efficiently.
tool for the routine purification of other GFP fusion proteins. Immunoaffinity chromatography has also been utilized in the purification of recombinant human TPB protein, with demonstrated activity observed by gel-shift and transcription assays [14]. Affinity chromatography is a powerful method to purify recombinant proteins from many genera. Recent research by Burgess and Thompson proposed an ingenious method for developing mAbs that were able to bind tightly, but release under gentle elution conditions (polyol-responsive mAb, PR-mAb), ideal properties for use in immunoaffinity chromatography [15]. In response to the rapidly growing field of proteomics, the use of recombinant proteins has also greatly increased in recent years [16]. Recombinant hybrids containing an affinity tag, to facilitate the purification of the target polypeptides or proteins are widely used. In this case, many different proteins, domains, or peptides can be fused with the GFP tag, not only as a monitoring probe, but also as an affinity tag. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (No. 30672370) and the National Key Technologies R&D Program of China.
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