Saudi Journal of Biological Sciences (2014) 21, 35–39
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
Recombinant expression and puriﬁcation of human placental growth factor 1 and speciﬁc camel heavy chain polyclonal antibody preparation Roghaye Arezumand a, Reza Mahdian a, Mahdi Behdani b, Hossein Khanahmad Jahangir Langari a, Nabiollah Namvarasl d, Reza Hassanzadeh-Ghasabeh e,f, Sirous Zeinali a,*
Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran Biotechnology Research Center, Venom & Biotherapeutics Molecules Lab., Pasteur Institute of Iran, Tehran, Iran c Department of Genetics and Molecular Biology, Medical School of Isfahan University of Medical Science, Isfahan, Iran d Department of Laboratory Animal Science, Research and Production Complex, Pasteur Institute of Iran, Karaj, Iran e Laboratory of Cellular and Molecular Immunology, Vrije University Brussel, Brussels, Belgium f Department of Structural Biology, NSF, VIB, Brussels, Belgium b
Received 14 February 2013; revised 18 April 2013; accepted 24 April 2013 Available online 7 May 2013
KEYWORDS PlGF; VHH; Polyclonal antibody; Angiogenesis; Heavy chain antibody
Abstract Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Unlike VEGF, PlGF is dispensable for normal cell development as well as playing various roles in pathological angiogenesis which occurs in tissue ischemia, inﬂammation, and malignancy. The PlGF-1 has been considered as a potential candidate for the diagnosis and targeting of pathological angiogenesis. Camelidae serum contains an important fraction of functional antibodies, called heavy-chain antibodies (HcAbs) that are naturally devoid of light chains. Camelid HcAbs recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody. Here, we describe the expression and puriﬁcation of recombinant human PlGF-1 (rhPlGF-1). This protein was subsequently used for the preparation of camel heavy chain polyclonal antibody against rhPlGF-1.
* Corresponding author. Address: Biotechnology Research Center, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran 1316543551, Iran. Tel./fax: +98 21 66480780. E-mail address: [email protected]
(S. Zeinali). Peer review under responsibility of King Saud University.
Production and hosting by Elsevier 1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2013.04.008
R. Arezumand et al. The recombinant expression plasmid pET-26b-hPlGF-1 was introduced into Escherichia coli BL21 cells to express the rhPlGF-1 protein. Puriﬁed rhPlGF-1 was used to immunize camel, the speciﬁc reactivity of HcAb was determined with ELISA and western blot. Western blot analysis indicated that the antiserum speciﬁcally reacted to the recombinant protein. The rhPlGF-1 protein and its antibody may be used for the development of detection assays needed for clinical research. ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
2. Materials and methods
Angiogenesis is an essential process in fetal development, tissue regeneration and remodeling in pathologic conditions such as tumor invasion and metastasis (Folkman, 2007). Angiogenesis is a complex process which is stimulated by a variety of angiogenic growth factors (Bikfalvi, 2012; Gordon et al., 2010). The vascular endothelial growth factors (VEGFs) are considered as key molecules in this process. The VEGF family currently includes VEGF-A, -B, -C, -D, -E, -F and placental growth factor (Roy et al., 2006). PlGF is a secreted protein that shares substantial structural similarity with VEGF. Four PlGF isoforms (i.e. PlGF-1–4) are produced by alternative splicing of PlGF genes in human. It has been shown that PlGF-1 can stimulate vessel growth and maturation directly by affecting endothelial and mural cells, and indirectly by recruiting pro-angiogenic cell types, the recruitment and maturation of angiogenesiscompetent myeloid progenitor cells and activating macrophages to release angiogenic and lymphangiogenic molecules (Selvaraj et al., 2003). In physiological condition the PlGF is undetectable but in pathological conditions is mainly up regulated. Therefore the blockade of the PlGF can lead to the inhibition of neovascularization and tumor metastasis (Fischer et al., 2008). Camelidae serum contains an important fraction of functional antibodies, called heavy-chain antibodies (HcAb) which are naturally devoid of light chains. Therefore, the Camelid HcAbs recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody (Behdani et al., 2012; Muyldermans et al., 2009). Nanobodies have several inherent, advantageous properties such as strict monomeric behavior, high afﬁnity, solubility and stability and remarkable expression yield of recombinant VHH in bacteria or yeast. These characteristics have made the VHH an attractive next-generation reagent for immunoassays and therapeutic applications. The aim of this study was the production of rhPlGF-1 and preparation of camel heavy chain polyclonal antibody directed against this protein.
2.1. Construction of pET-26b-hPlGF-1 plasmid
Escherichia coli B21 strain and pET-26b plasmid were purchased from Iranian Gene Bank (Pasteur Institute of Iran, Tehran, Iran). The synthetic hPlGF-1 construct (BIOMATIK, Canada) was composed of coding sequence of the human PlGF-1 inserted between NcoI and XhoI restriction sites of the pET26b plasmid (Fig. 1). This construct was transformed into E. coli BL21 cells. The integrity of the ﬁnal construct (pET26b-hPlGF-1) and the transformation process were conﬁrmed by restriction digestion and DNA sequencing. 2.2. Expression and puriﬁcation of rhPlGF-1 The protein expression was induced by the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG). After overnight incubation at 37 C, the bacterial cells were pelleted and homogenized by ultrasonication in analysis buffer (8 M urea, 20 mM Tris–HCl, 500 mM NaCl, 50 mM Imidazol, 0.5% triton X-100 and protease inhibitor). The bacterial cell lysate was centrifuged at 8000g for 30 min. The supernatant was loaded on a Ni–NTA column and washed with 4 M urea, 20 mM Tris–HCl, 500 mM NaCl, 60 mM Imidazol, and protease inhibitor. The bound proteins were eluted with 500 mM Imidazole in PBS. The eluted protein was loaded on a gel ﬁltration column (Sephadex 75- GE-Healthcare). 2.3. SDS–PAGE and western blotting analysis Puriﬁed rhPlGF-1was determined on SDS–PAGE with 12% resolving gel and 5% stacking gel. For western blotting the separated protein was transferred onto nitrocellulose membrane, and the membrane was blocked with 2% skimmed milk in PBS for 2 h at room temperature (RT) and incubated with 1/3000 mouse anti-hPlGF-1(Cell Science) overnight at 4 C.
(a) Schematic expression cassette of pET26-hPLGF-1, (b) The amino acid sequence of hPLGF-1.
Recombinant expression and puriﬁcation of human placental growth factor 1 and speciﬁc camel heavy The membrane was subsequently incubated with 1/10,000 horseradish peroxidase (HRP)-conjugated anti-mouse IgG overnight at 4 C. The immunoreactive bands were visualized with developer solution containing 4-choloro-1-naftol. The commercial human PlGF (Cell Science) was used as control. A similar set up was used to determine the immunoreactivity and speciﬁcity of the camel polyclonal antibody raised against the recombinant protein. Brieﬂy, the rhPlGF-1 protein on the membrane was incubated with the 1/1000 dilution of puriﬁed camel heavy-chain fraction overnight at 4 C. The membrane was then incubated with 1/16,000 rabbit anti-camel antibody for 1 h at RT. Then, the membrane was incubated with 1:3000 HRP-conjugated anti-rabbit to reveal immuno reactive bands. 2.4. Preparation of polyclonal camel heavy chain antibody against rhPlGF-1 An 8-month-old male camel Research and Production Complex was kept at Pasteur Institute of Iran (Animal Sciences Branch) under appropriate conditions. Blood serum samples were collected before the injection of the antigen and stored at 80 C. The camel was immunized with six weekly subcutaneous injections at neck. At each injection, 100 lg of rhPlGF-1 in 2 ml PBS was mixed with 2 ml Freund’s complete adjuvant for the ﬁrst immunization, and with 2 ml of Freund’s incomplete adjuvant for the following immunizations. The serum samples were harvested at weeks 4 and 7. Heavy chain antibodies were isolated according to the standard protocol (Hamers-Casterman et al., 1993). Brieﬂy, 5 ml of camel serum was loaded on protein G Sepharose column (GEHealthcare) and was washed with 20 mM phosphate buffer pH 7. IgG3 fraction was eluted with 0.15 M NaCl and 0.58% acetic acid (pH 3.5) and IgG1 fraction was eluted with 0.1 M Glycin–HCl (pH 2.7). For the isolation of IgG2 fraction, the ﬂow through of the protein G Sepharose column was applied onto protein A Sepharose column (GE-Healthcare). After
washing with phosphate buffer, the absorbed fraction was eluted with 0.15 M NaCl and 0.58% acetic acid (pH 4.5). 2.5. Functional assay The reactivity of the puriﬁed camel heavy chain antibody with recombinant rhPlGF-1 was examined by western blotting, as described above and ELISA. For ELISA each well was coated with 1 lg/ml of rhPlGF-1 and blocked with 2% skimmed milk. The puriﬁed heavy chain polyclonal antibody was added to wells at different dilutions starting from 1/200 to 1/50,000. The bound antibody was detected using 1/16,000 dilution of rabbit anti-camel antibody and 1/3000 dilution of HRP-conjugated anti-rabbit antibody. 3. Results 3.1. Expression and characterization of rhPlGF-1 The integrity of the pET-26b-hPlGF-1 construct was shown by double digestion with NcoI and XhoI restriction enzymes and DNA sequencing. The rhPlGF-1was expressed as His-tag fusions and puriﬁed by Ni–NTA afﬁnity chromatography and gel ﬁltration. The gel ﬁltration result revealed that the recombinant protein is monomeric (Fig. 2). The ﬁnal yield was 5 mg/lit. The puriﬁcation process was conﬁrmed using SDS–PAGE. The rhPlGF-1 is present as a single band about 15 kDa (Fig. 3a). The western blot analysis was done with commercial anti-PlGF antibody (Cell Science) and revealed that the puriﬁed recombinant protein was successfully transferred onto the nitrocellulosemembrane and had migrated at estimated size (Fig. 3b). 3.2. The camel HcAb speciﬁc to rhPlGF-1 The camel was immunized weekly for six times with rhPlGF-1. The immune response was followed by ELISA analysis with
Gel ﬁltration chromatography proﬁle of the Ni–NTA puriﬁed rhPLGF-1, loaded onto a superdex 75 column in PBS buffer.
R. Arezumand et al.
Figure 3 Expression and characterization of the rhPlGF-1. (a) SDS–PAGE. Lane1, before induction; Lane2, Induced with 1 mM IPTG; Lane3, Puriﬁed protein. (b) Western Blotting. Lane 1, puriﬁed rhPlGF-1 protein; Lane 2, commercial human PlGF-1. Lane M: protein molecular weight marker.
After week 7
After week 4
1.2 1 0.8 0.6 0.4 0.2 0
Figure 5 Sensitivity analysis of HcAbs by ELISA. The camelid HcAbs antibody at different dilutions (1:200 to 1:50,000) reacted with the recombinant rhPLGF protein.
Figure 4 The SDS–PAGE pattern of puriﬁed camel antibodies in reduced condition. Lane 1, the conventional camel antibody (IgG1) with light and heavy chain. Lane 2, 3, the heavy chain antibodies (IgG2, 3 respectively).
puriﬁed HcAb. The standard protocol was used for HcAbs puriﬁcation. As shown in Fig. 4, we can separate the HcAbs from conventional antibodies. During the course of immune response, the puriﬁed HcAbs were diluted at 1/200 to 1/50,000 and tested for reactivity. As
shown in Fig. 5, speciﬁc antibody titers raised rapidly after the fourth injection. Western blot analysis was performed to determine the speciﬁcity of the heavy-chain antibodies (Fig. 6), which revealed that the rhPlGF-1 could be recognized by the heavy-chain polyclonal antibody. These results indicated that the heavychain polyclonal antibody has high speciﬁcity for the rhPlGF-1 protein. 4. Discussion Development of antibodies speciﬁc to biomarkers has mainly focused on mouse antibodies. Alternatively, recent studies on camelid immunoglobulins provide a compelling rationale for
Recombinant expression and puriﬁcation of human placental growth factor 1 and speciﬁc camel heavy
Acknowledgments This work was ﬁnancially supported by Iran National Science Foundation (Grant No. 89002262) and Pasteur Institute of Iran. References
Figure 6 Determination of speciﬁcity by western blot analysis in non-reducing condition with Heavy-chain polyclonal antibody. Lane 1, puriﬁed rhPlGF-1, Lane 2, commercial human PlGF-1, M, protein molecular weight marker.
the development of antibody-based approaches (e.g. the detection of the tumor-associated markers). In this study, we described the expression and puriﬁcation of rhPlGF-1 and its application for camelid polyclonal antibody preparation. The pET26b-hPlGF-1 construct expressed more than 5 mg/lit of the desired protein and the puriﬁed product was successfully used for camel immunization. The camel heavy chain polyclonal anti-PlGF-1 detected the rhPlGF-1 protein as well as commercial rhPlGF-1in an ELISA assay. Interestingly, over 50% of camelid immunoglobulin lack light chain and the 15 kDa antigen binding domain of the heavy chain antibody (VHH, obtained by protease cleavage) is considerably smaller than papain cleaved Fab fragments (60 kDa) of conventional IgG (Rahbarizadeh et al., 2011). Camelid heavy chain IgG is also less immunogenic than most mammalian IgG (Cortez-Retamozo et al., 2002) indicating that intravenous administration of camelid antibody is less likely to induce the anti-antibody and anaphylactic adverse reactions (Lalloo and Theakston, 2003). Unlike conventional IgG, in some reports, the antigen-binding capabilities of camelid HcAbs and derived VHH are not affected by exposure to high temperatures. (Graef et al., 2011; Ladenson et al., 2006; van der Linden et al., 2000). PlGF-1 is a tumor associated marker which has several important roles including role in pathological conditions in tumor angiogenesis, as an important bio marker in patients with coronary artery disease, facilitates cardiac healing after myocardial hypoxia/ischemia, and improves wound healing in diabetes (Ribatti, 2011). Therefore, PlGF protein level detection is a useful tool for disease progress determination. Our results showed that the anti-PlGF-1 polyclonal HcAb can be used to detect the PlGF-1 and for the development of diagnostic assays. Furthermore, the ﬁndings reported here may pave the way for the development of a speciﬁc Nanobody against PlGF for in vivo studies and anticancer applications.
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