Prokaryotic expression, purification, and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyltransferase 14

Protein Expression and Purification 56 (2007) 1–7 www.elsevier.com/locate/yprep

Prokaryotic expression, purification, and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyltransferase 14 Chen Wu a

a,b

, YuanYuan Wang a, MinJi Zou a, YaoJun Shan b, GuangYin Yao b, Ping Wei c, GuangYu Chen a, JiaXi Wang a, DongGang Xu a,*

Laboratory of Molecular Genetics, Beijing Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, China b College of Life Sciences, Hebei University, Baoding 071002, China c Department of Endocrine, Southwest Hospital, Third Military University, Chongqing 400038, China Received 1 April 2007, and in revised form 18 April 2007 Available online 24 May 2007

Abstract Polypeptide N-acetylgalactosaminyltransferase 14 (GalNAc-T14, EC 2.4.1.41) belongs to a large subfamily of glycosyltransferases residing in the Golgi apparatus. N-Acetylgalactosaminyltransferases (GalNAc-Tases) catalyze the first step in the O-glycosylation of mammalian proteins by transferring N-acetyl-D-galactosamine (GalNAc) to peptide substrates. Here, the cloning, expression, purification, and polyclonal antibody preparation of GalNAc-T14 were described. A full-length GalNAc-T14 cDNA was inserted in a prokaryotic expression plasmid pGEX-4T-1 at the EcoRI and XhoI restriction sites. pGEX-4T-T14 was highly expressed in Escherichia coli (E. coli) BL21(DE3) cells after induced by isopropyl-b-D-thiogalactoside (IPTG). The expressed GST-GalNAc-T14 fusion protein was purified by GSTrap FF chromatography and then used as antigen to immunize rabbits. The obtained antiserum was precipitated by 50% saturated ammonium sulfate and then purified by DEAE–Sepharose FF chromatography. To confirm the activity and specificity of the GalNAc-T14 antibody, we constructed the plasmid pFLAG-GalNAc-T14 to transfect transiently HEK 293T cells. Transiently expressed FLAG-GalNAc-T14 was identified by Western blot analysis with GalNAc-T14 antibody and FLAG monoclonal antibody, respectively. The production of the polyclonal antibody against GalNAc-T14 provides a good tool for studying the biofunctions of GalNAc-T14. Ó 2007 Elsevier Inc. All rights reserved. Keywords: GalNAc-T14; Prokaryotic expression; Purification; Polyclonal antibody

Systematic functional analysis of posttranslational modifications, such as glycosylation, is getting more and more important. It has been suggested that the diversity of O-linked oligosaccharides on glycoproteins play roles in protection from proteolytic degradation, alteration of substrate structural conformation, lymphocyte trafficking, sperm–egg binding, and tumor cell adhesion [1–5]. The biosynthesis of O-linked oligosaccharides is determined by the repertoire of glycosyltransferases residing in the Golgi

*

Corresponding author. Fax: +86 010 66931380. E-mail address: [email protected] (D. Xu).

1046-5928/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2007.04.027

apparatus. N-Acetylgalactosaminyltransferase (GalNAcTase, EC2.4.1.41) is a glycosyltransferase involved in the first step of O-glycan synthesis [6]. It catalyses the transference of GalNAc group from UDP-GalNAc to serine or threonine residues on target proteins to form a GalNAc a-O glycosidic linkage. GalNAc-Tase activity was first reported 39 years ago by McGuire and Roseman [7]. To date, 15 GalNAc-Tases (GalNAc-T1-T15) have been identified in mammals, and functional profiles of each member of the family have been characterized, showing that these enzymes have not only different substrate specificities, but also specific tissue expression patterns, which are related to the diverse

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functions of these different subtypes of GalNAc-Tase [8– 10]. GalNAc-T14 was first reported in 2003 by Wang et al. who cloned its cDNA and designated it as GalNAc-T14 and confirmed that GalNAc-T14 is a new member of the GalNAc-Tase family. Quantitative real time PCR analysis revealed that GalNAc-T14 transcript was highly expressed in kidney and it suggested that GalNAcT14 might be involved in the O-glycosylation in kidney [10]. O-Glycosylation is an important posttranslational modification, and the GalNAc-Tase family is closely related to invasion, metastasis and proliferation of many cancer cells [6]. So investigation of the GalNAc-Tase family may bring new insight into cancer research. Since GalNAc-T14 is a new member of GalNAc-Tase family, little is known about its regulation at protein level and its biological significance. Lacking of commercial antibody against GalNAc-T14, studies on biofunctions related to GalNAc-T14 are limited. In this paper, the cloning and expression of human GalNAc-T14 gene in Escherichia coli (E. coli)1, purification of recombinant proteins, and generation of polyclonal antibody against GalNAc-T14 are described. The prepared antibody can be useful for the study of expression and distribution of GalNAc-T14 in various tissues at protein level and to elucidate its biofunctions and regulation mechanism in cancer.

72 °C for 2 min, and then final extension at 72 °C for 7 min. The amplified GalNAc-T14 gene was gel-purified by the Gel Extraction Kit (Omega Biotechnology Co., USA). After digestion with EcoRI and XhoI, the purified product was inserted into the corresponding region of pGEX-4T-1 expression vector and confirmed by restriction analysis and sequencing. The correct recombinant prokaryotic expression vector was named as pGEX-4T-T14.

Materials and methods

Extraction of GST fusion proteins

Materials

The cells were harvested by centrifugation at 10,000 rpm for 10 min at 4 °C. The pellet was suspended (10 ml/g wet weight) in lysis buffer (PBS, pH 7.3, 1.0 mM EDTA, 1.0 mM PMSF and 1.0 mg/ml lysozyme). The suspension was incubated for 20 min at 4 °C with stirring. Following sonication, the suspension was centrifuged at 12,000 rpm for 15 min. The clear supernatant (soluble fraction) was collected and the remaining pellet (insoluble fraction) containing inclusion bodies was resuspensed in an equal volume of lysis buffer. Both soluble and insoluble fractions were then analyzed on 12% SDS–PAGE.

pGEX-4T-1 plasmid was purchased from Amersham Biosciences, USA, while all restriction enzymes and the Expand High Fidelity PCR system were purchased from TakaRa (Japan). All PCR products used for cloning were confrmed by sequencing at Invitrogen Biotechnology Co., Ltd (Shanghai, China). Cloning of GalNAc-T14 and constructions of expression vector A full-length cDNA of GalNAc-T14 (GenBank Accession No. NM_024572) was amplified from a cDNA library of human brain using the Expand High Fidelity PCR system with a pair of gene specific primers (forward: 5 0 -gga attccggcgcctgactcgtcg-3 0 , reverse: 5 0 -ccgctcgagtcaagagctca ccatgtcccagtgctgg-3 0 ) containing the EcoRI and XhoI restriction sites, respectively. The reaction was carried out with the following procedures in a GeneAmp PCR System (Perkin Elmer, USA): initial denaturation at 95 °C for 5 min followed by 30 consecutive cycles of denaturation at 95 °C for 30 s, annealing for 30 s at 60 °C, extension at 1 Abbreviations used: E. coli, Escherichia coli; LB, Luria–Bertani; OD600, optical density; IPTG, isopropyl-b-D-thiogalactopyranoside; ELISA, enzyme-linked immunosorbent analysis; RCC, renal cell carcinomatous; IGFBP-3, Insulin-like growth factor binding protein 3.

Expression of recombinant protein in E. coli Escherichia coli BL21 (DE3) cells were transformed with recombinant plasmid, pGEX-4T-T14. To obtain as much soluble fusion protein as possible, we optimized the condition for induction. The GST fusion protein was expressed on a large scale as follows. The engineered bacteria were cultured in 25 ml Luria–Bertani (LB) liquid medium containing ampicillin (100 lg/ml) and grown overnight at 37 °C and 220 rpm, then transferred to 500 ml of fresh medium and incubated for another 2 h until the optical density (OD600) of the cultured cells reached 0.6. Expression of the fusion protein was induced with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 20 °C overnight. The recombinant protein was analyzed by SDS–PAGE, while protein extracts of cells transformed with the empty vector pGEX-4T-1 were used as the control.

Purification of GST fusion protein GST fusion protein was purified by GSTrap-FF affinity chromatography (Amersham Biosciences AB, Uppsala, Sweden) according to the manufacturer’s protocol. Briefly, the column was equilibrated with 5 column volumes of binding buffer (PBS pH 7.3, 1.0 mM DTT). The sample was loaded onto the column at a flow rate of 0.2–1 ml/ min, and the bound protein was eluted by adding 5 column volumes of elution buffer (50 mM Tris–HCl, 10 mM reduced glutathione, pH 8.0, a flow rate 1–2 ml/min). The eluted protein was carefully collected and analyzed by 12% SDS–PAGE. The purified protein was identified by Western blotting using anti-GST polyclonal antibody (Sigma) and the concentration of the GST fusion protein was tested by Lowry method.

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Production and purification of polyclonal antibodies against the recombinant protein

sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4) containing proteinase inhibitors.

The purified recombinant protein was used for preparing antibodies in New Zealand white rabbits. The rabbit was first immunized subcutaneously with 500 lg recombinant protein in Freund’s complete adjuvant. Two booster injections were given with 250 lg recombinant protein each in incomplete Freund’s adjuvant at 3-weeks interval, and the antiserum was collected 7 days after the last boost. The rabbit IgG fraction was precipitated from the immune serum with 50% saturated (NH4)2SO4 and purified by DEAE–Sepharose column chromatography, and then the antibody titer was determined by ELISA.

Identification of GalNAc-T14-transfected 293T cells by Western blot

Antiserum titer determination by ELISA Antibody titer was measured using an indirect enzymelinked immunosorbent analysis (ELISA). The purified antigen, diluted to 10 lg/ml in 50 mM carbonate salt buffer (pH 9.6), was coated on plates at 100 ll aliquot per well, 4 °C overnight. The wells were washed three times with PBS–Tween buffer (0.05% Tween 20 in PBS). The coated wells were blocked with 200 ll of 3% BSA for 1 h at 37 °C and then incubated with 150 ll polyclonal antibodies against GalNAc-T14 with different dilutions (from 1:1000 to 1:25600). After incubation for 2 h at 37 °C, the wells were incubated with 150 ll horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution 1:5000, Sigma, USA) for 1 h at 37 °C after thorough washing. Peroxidase activity on the immunoplate was detected using o-phenylenediamine and H2O2 as enzyme substrates. Color development was stopped with 2M of H2SO4 and the absorbance was measured at 490 nm using Microplate Reader (Bio-Rad, USA). Cell culture and transfection HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, New York) supplemented with 10% FCS (HyClone, Hampton, NH), 2 mM Lglutamine, 20 U/ml penicillin, and 20 mg/ml streptomycin. The culture was kept in an incubator with 5% CO2 and 95% humidified air at 37 °C. Cells were seeded in a 6-well culture plate, and then grown to 50–70% confluence. Cells were washed with phosphate-buffered saline, and 1.5 ml serum-free medium was added to each well. pFLAG-CMV2 (Sigma) with or without a full-length human GalNAc-T14 cDNA was transfected transiently into HEK 293T cells, respectively, using Lipofectamine2000 (Invitrogen) according to manufacture’s instructions. After incubation for 6 h, each dish was replaced with 2 ml complete medium and incubated for 48 h. The cells were harvested, and then lysed in cell lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM

Western blotting was performed according to the standard procedure. After 12% SDS–PAGE, the gel was immersed in the transfer buffer (48 mM Tris–HCl, 39 mM glycine, and 20% methanol, pH 9.2), and the proteins were transferred to NC membrane. The membrane was incubated for 1 h with blocking buffer (5% BSA in TBS) at RT. After being washed three times (10 min each) with TBS–Tween buffer, the membrane was incubated with the anti-GalNAc-T14 (1:1000) or anti-FLAG (1:1500, Sigma) polyclonal antibody for 2 h at RT. The membrane was then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Sigma) at RT after thorough washing. The membrane was washed as described above and then analyzed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Canada) and exposed to Kodak BioMax X-ray film (Eastman Kodak Co., Rochester, NY) for 2–5 min. Results and discussion Cloning cDNA for GalNAc-T14 and construction of its expression vector The complete cDNA of GalNAc-T14 was amplified by PCR from a cDNA library of human brain. Gene specific primers were designed according to the published sequence of human GalNAc-T14. EcoRI and XhoI sites were designed in the primers to facilitate cloning in pGEX-4T-1 (Fig. 1a). The PCR product was ligated to pGEX-4T-1. The recombinant plasmid pGEX-4T-T14 was identified by restriction analysis (Fig. 1b) and then confirmed by sequencing. Expression of recombinant protein and identification by Western blot The confirmed construct pGEX-4T-T14 and pGEX4T-1 were transformed into E. coli BL21(DE3) cells, respectively. Expression of the fusion protein was induced at 20 °C and predicted to encode a recombinant protein with a molecular weight of 87 kDa. To examine the distribution of expressed recombinant protein in soluble and insoluble fractions, both the supernatant and pellet of cell lysate after sonication were analyzed. Samples from BL21 carrying pGEX-4T-T14 or pGEX-4T-1 were analyzed by SDS–PAGE and subsequent Coomassie brilliant Blue staining. Bands of GST-GalNAc-T14 with expected M.W. of 87 kDa and GST with expected M.W. of 26 kDa were observed (Fig. 2a). The soluble GST-GalNAc-T14 in supernatant is pointed by an arrow (lane 5).

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Fig. 1. Construction of pGEX-4T-T14. (a) Schematic diagram of the ORF of GalNAc-T14 inserted in pGEX-4T-1 expression vector. (b) Identification of recombinant plasmid pGEX-4T-T14 by restriction analysis. Lane 1, pGEX-4T-T14/EcoRI and XhoI; lane 2, pGEX-4T-1/EcoRI and XhoI; M, DNA marker.

The recombinant protein was also analyzed by Western blot using anti-GST monoclonal antibody. The predicted bands of 87 kDa recombinant GST-GalNAc-T14 protein (lane 1) and 26 kDa GST protein (lane 2) were visualized as shown in (Fig. 2b).

and loaded to a column. The tagged proteins bound to the GSTrap FF affinity column were eluted with elution buffer. The purified protein was dialyzed and quantified to be 253 lg/mL by Lowry method. Titer and specificity analysis by ELISA and Western blot

Purification of the fusion protein Purified GST-tagged protein is shown in Fig. 3, lane 1. The GST fusion protein was found in two fractions, soluble and insoluble. There was more insoluble than soluble protein, but the soluble protein can be purified in its native form. So, sufficient soluble fusion proteins were prepared

The IgG fraction against GalNAc-T14 was purified from rabbit antiserum by (NH4)2SO4 precipitation and DEAE–Sepharose chromatography. The titer of the obtained IgG against GalNAc-T14 was determined by ELISA. The antibody at different dilutions (1000- to 256,000-fold) was reacted with an equal amount of the

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Fig. 2. Expression of GST fusion proteins (a) and their assays with Western blotting (b). (a) Expression of GST-GalNAc-T14 analyzed by 12% SDS– PAGE. Lane 1,whole lysate of cells harboring pGEX-4T-1 without IPTG induction; lane 2, whole lysate of cells harboring pGEX-4T-1 after IPTG induction; lane 3, whole lysate of cells harboring pGEX-4T-T14 without IPTG treatment during induction; lane 4, whole lysate of cells harboring pGEX4T-T14 after induction with IPTG; lane 5, lysates of soluble fraction of cells harboring pGEX-4T-T14 after induction with IPTG; lane 6, lysates of insoluble fraction of cells harboring pGEX-4T-T14 after induction with IPTG; M, protein molecular mass standards. (b) Identification by Western blotting using anti-GST monoclonal antibody. Lane 1, GST-GalNAc-T14; lane 2, GST.

fusion protein (1 lg). The pre-immunized rabbit serum was used as the negative control. The antibody titer is defined as the highest dilution of serum at which the A490 ratio (A490 of postimmunization sera/A490 of preimmunization sera) is greater than 2.1. The values were the means ± SE (n = D3). The antibody titer was found to be approximately 1:16,000. The activity and specificity of the purified antibody was further tested by Western blotting analysis. FLAG-GalNAc-T14 was expressed transiently by transfecting the pFLAG-CMV-T14 plasmid into HEK 293T cells. The lysis of cells harboring recombinant FLAG-GalNAc-T14 was loaded as the target and the lysis of the cells transfected with pFLAG-CMV2 was used as the negative control. Western blotting was performed with the purified anti-GalNAc-T14 antibody and anti-FLAG antibody at a dilution of 1:1000 and 1:1500, respectively. The result of Western

blotting with the two antibodies is shown in Fig. 4. An expected 61 kDa band of FLAG-GalNAc-T14 was detected in whole lysis of 293T cell transfected with pFLAG-GalNAc-T14 (Fig. 4a). There was a smaller band (1 kDa) in the lysis of 293T cell transfected with pFLAGCMV2 using anti-FLAG antibody (data not shown). Western blotting analysis with the polyclonal antibody against the GalNAc-T14 fusion protein displayed a 61 kDa band in both lyses (Fig. 4b). The same samples were also incubated with the pre-immune serum but no reaction was observed. The result indicated that 293T cells express endogenously GalNAc-T14. It is also showed that GalNAc-T14 is expressed endogenously in human renal cell carcinomatous (RCC) cell lines 786O, but not in human breast cancer cell lines MCF-7 (unpublished), which is coincident with quantitative analysis of the pp-GalNAcT14 transcript in human tissues by real-time PCR [10].

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The purified antibody can recongnize the GalNAc-T14 with high activity and specificity, and serve as a good tool for further research the biofunctions related to GalNAcT14 protein.

colorectal carcinoma. Strong expression of GalNAc-T3 was associated with a strong likelihood of survival [18]. GalNAc-T14 has been confirmed as a new member of the GalNAc-Tase family and a ubiquitous protein present in various tissues. GalNAc-T14 gene was mapped to the human chromosome 2 at p23.2 and contains an ORF of 1659-base pairs that encoding a 552-amino acid type-II membrane protein that consists of a N-terminal cytoplasmic domain, followed by a transmembrane domain, a stem region, and a catalytic domain. The catalytic domain contains a GT1 motif, a Gal/GalNAc transferase motif, and a ricin-like lectin motif, which are commonly observed in the GalNAc-Tase family proteins [19]. GalNAc-T14 shares 49.9% amino acid sequence homology with pp-GalNAcT2 and forms a subfamily with GalNAc-T2 on the phylogenetic tree [10]. To date, very few functional studies on GalNAc-T14 have been carried out, which is mainly due to the unavailablility of a commercial anti-GalNAc-T14 antibody available. The purpose of the present study is to generate the antibody for further studying the biofunctions of the GalNAc-T14 protein. In order to obtain abundant target protein to generate polyclonal antibodies against GalNAc-T14, prokaryotic expression system was used. GalNAc-T14 fusion protein was highly and rapidly expressed in E. coli, which made the production of antibody against GalNAc-T14 more convenient. In conclusion, in the present work a full-length GalNAc-T14 cDNA from cDNA library of human brain was cloned and highly expressed in E. coli, and a highly specific and sensitive antibody against GalNAc-T14 was produced. The antibody will serve as a tool to explore the function of GalNAc-T14, such as identifying its potential binding partners. We recently found a new binding partner of GalNAc-T14—insulin-like growth factor binding protein 3 (IGFBP-3) [20]. The prepared antibody would be useful for further study of their interaction. The antibody may also contribute to the study of GalNAc-T14’s structure and the mechanism of the pathogenesis and progression of cancer cells. Such results will provide a substantial base for further clarification of the GalNAc-T14 function.

Conclusion

Acknowledgment

O-glycans impart unique structural features to mucin glycoproteins and numerous membrane receptors [11,12]. Structure-function studies have demonstrated that O-glycans also impart resistance to thermal change and proteolytic attack in a number of diverse proteins, such as glucoamylase [13] and apolipoprotein A [14]. The expression of the Tn antigen in cancer cells could be the result of a deregulation of glycosyltransferases (e.g., changes in enzyme activity and/or in substrate specificity) [15]. The expression of the GalNAc-Tase family in cancer cell lines and malignant tissues have been examined in several studies [16,17]. It was concluded that expression of GalNAc-T3 is a useful indicator of prognosis in patients with

This study was financially supported by National Natural Science Foundation of China (No. 30270634).

Fig. 3. Purification of GST-GalNAc-T14. Lane 1, purified recombinant protein; M, protein molecular mass standards.

Fig. 4. Western blot analysis of the activity and specificity of antisera. (a) Western blot with anti-FLAG monoclonal antibody. (b) Western blot with anti-GalNAc-T14 antibody. Lane 1, lysis of 293T cells transfected by pFLAG-CMV2; lane 2, lysis of 293T cells transfected by pFLAG-CMVGalNAc-T14.

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