Expression and Production of Pigment Epithelium-derived Factor (PEDF) and PEDF Receptor Variants from Mammalian and Bacterial Cells

Abstract

Human SERPINF1 gene codes for pigment epithelium-derived factor (PEDF), a secreted glycoprotein and member of the SERPIN superfamily. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. The cells produced and secreted recombinant PEDF proteins into the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. PEDF has affinity for PEDF-receptor (PEDF-R), a membrane-linked lipase encoded by the PNPLA2 gene. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3’end deletions and by induction with isopropyl β- d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. C-terminally truncated PEDF-R versions containing the ligand binding region retained the ability to bind PEDF. The data demonstrate that mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for further use in structural and biological studies.

1. Introduction

The human SERPINF1 gene codes for the pigment epithelium-derived factor (PEDF) polypeptide of 418-amino acids [1]. Mammalian cells expressing the SERPINF1 gene produce and secrete a mature glycosylated PEDF protein with an apparent molecular weight of 50,000 and without the 20 amino acids from its amino-terminal end that correspond to the secretion signal peptide [2]. As a member of the serine protease inhibitor (SERPIN) superfamily, the 3D structure of PEDF has homology to serpins; however, it lacks demonstrable inhibitory activity against serine proteases, placing it in the non-inhibitory subgroup of serpins [3]. This serpin might have lost its inhibitory property during evolution and gained several biological activities. The first biological activity to be reported was neurite-outgrowth activity on retinoblastoma cells by PEDF protein found in the culturing medium of human fetal retinal pigment epithelial (RPE) cells [4]. Later, it was demonstrated that PEDF also protects neuronal cells, photoreceptors, and other retinal neuronal cells from death in vitro and in vivo, and in addition possesses anti-angiogenic, anti-tumorigenic and anti-inflammatory properties [2], [5], [6]. PEDF is being investigated as a therapeutic candidate for treatment of conditions as retinal degenerations, ocular neovascularization, heart disease, and cancer [5], [6].

Several functional regions have been mapped to distinct areas in the PEDF polypeptide. PEDF affinity for extracellular matrix components such as collagens and glycosaminoglycans requires specific amino acids of its sequence. The negatively charged aspartic amino acids at positions D268, D258, and D300 of PEDF (human sequence numbering) are required for collagen binding [7], while the positively charged amino acids R146, K147, and R149 are crucial for heparin binding, and K189A/K191A/R194A/K197A forms a hyaluronan binding motif [8]. The 34-mer amino acid region spanning positions 44–77 of human PEDF confers anti-angiogenic properties [9]–[11], and the 44-mer amino acid region (positions 78–121) confers neurotrophic activities similar to the full-length protein [12]–[16].

The biological effects of PEDF are mediated by interactions with cell surface receptors [17]. One of these receptors identified in our laboratory is PEDF-R, a membrane-linked lipase with affinity for PEDF [18]. The gene that codes for human PEDF-R is PNPLA2 and has an open reading frame for a polypeptide of 504 amino acid amino acid residues [18]. The polypeptide sequence contains a patatin phospholipase-like region with a catalytic site consisting of a serine (position 47) and aspartic acid residue (position 166) [19], [20] and exhibits phospholipase A2, triglyceride lipase, and transacylase enzymatic activities [18], [21]–[23]. PEDF binding to PEDF-R enhances its phospholipase A2 activity to release fatty acids from phospholipids, such as docosahexaenoic acid (DHA), an omega-3 fatty acid that is a primary structural component of the mammalian retina, as well as of the cornea, brain, and cerebral cortex [18], [24]. The free DHA can extrude the intracellular calcium accumulated in photoreceptor cells of degenerating retinas interfering with signaling cascades to prevent death [25]. The binding to PEDF-R is required to mediate the neurotrophic responses of PEDF [26]. The human PEDF-R polypeptide sequence has determinants for PEDF binding within an ectodomain spanning amino acid positions 203–232 that are critical for enzymatic stimulation [26]. The region of PEDF composed of 17 residues (positions 98–114) binds and activates PEDF-R, like the full-length PEDF [13]. Interestingly, an H105A alteration of PEDF enhances PEDF-R receptor binding, while an R99A alteration abolishes PEDF-R binding, and in turn, cytoprotection activity in vitro and in photoreceptors of the rd1 and rd10 mouse models of retinal degeneration in vivo [13], [27]. These findings point to an important role played by the PEDF/PEDF-R complex in neurotrophic promotion. Studies to enhance our understanding of the interaction between PEDF and PEDF-R are needed.

Efficient production of recombinant PEDF and PEDF-R proteins is a pre-requisite for in vitro modeling of their interactions, as they are involved in neurotrophic activity in vivo. Although expression vectors for both human PEDF and PEDF-R have previously been constructed, expression and production of variants for these proteins have yet to be established and optimized to obtain high yields. In this study, we developed protocols to express and purify large amounts of PEDF and of PEDF-R using respectively a mammalian and a bacterial expression system, and discuss their use in structural and biological studies.

2. Materials and Methods

2.1. Construction of pCEP4 plasmids

The cDNA sequences used for the expression vectors were for those encoding for PEDF[R99A], PEDF[H105A], PEDF[D256A, D258A, D300A], and PEDF[K146A, K147A, R149A]. The construction of expression vectors is illustrated in Figure S1. In this study, PEDF[D256A, D258A, D300A] is referred to as PEDF[Collagen], and PEDF[K146A, K147A, R149A] is referred to as PEDF[Heparin]. The cDNA of PEDF versions in the pBK-CMV vector (Stratagene), termed pBK-PEDF, pPEDF(K146A/K147A/R149A) and pPEDF(D256A/D258A/D300A) [8], as well as the cDNA of PEDF[R99A] and PEDF[H105A] were removed from the pBK donor vectors. For this purpose, 5 μg of each plasmid DNA were incubated with 5 units of EcoRI (New England BioLabs) in NEBuffer^™ 2.1 (New England BioLabs) at 37°C for 3 h to linearize the plasmid. The linearized DNA mixtures were incubated with a Quick Blunting^™ Kit, which included a T4 DNA Polymerase (New England BioLabs) according to manufacturer’s instructions, at room temperature for 20 min to remove the 3’ overhangs at the end the DNA. The reaction was stopped by incubation at 70°C for 10 min. Then, 40 units of HindIII (New England BioLabs) were added to the blunt-ended DNA mixtures and incubated at 37°C for 3 h to remove the PEDF cDNA fragment from the DNA, which was 1.5kb in length and flanked by EcoRI-blunt and HindIII ends. The total volume of each of the reactions (52 μl) was loaded onto an 0.8% agarose gel in 1X TAE buffer (40 mM Tris, 20 mM acetic acid, 1mM EDTA, Thermo Fisher Scientific) for resolution of the DNA fragments by electrophoresis. The bands of 1.5kb corresponding to PEDF cDNAs were excised from the gel and purified using the QIAquick® Gel Extraction Kit (Qiagen) and the MinElute® columns (Qiagen) following the manufacturer’s instructions. Elution was in 15 μl of elution buffer (10 mM Tris-Cl, pH 8.5). DNA concentration was determined using the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific).

To prepare the receiving vectors, 5 μg of pCEP4 DNA (Thermo Fisher Scientific) were digested with 5 units of KpnI (New England BioLabs) in NEBuffer^™ 1.1 (New England BioLabs) and incubated at 37°C for 3 h. The 3’ overhang ends of the linearized pCEP4-KpnI DNA were removed by incubation with T4 DNA Polymerase at room temperature for 20 min. The reaction was stopped by incubation at 70°C for 10 min to denature the enzymes. The pCEP4-KpnI DNA was purified using the QIAquick® PCR Purification Kit (Qiagen) according to manufacturer’s instructions and eluted in 40 μl of elution buffer. The pCEP4/KpnI-blunt DNA was digested with 20 units of HindIII in NEBuffer^™ 2.1 (New England BioLabs) incubated at 37°C for 2 h. Then, 10 units of alkaline phosphatase, calf intestinal (CIP) (New England BioLabs) were added to the mixture and incubated at 37°C for 1 h. The total volume of the reaction (52 μl) was loaded onto an 0.8% agarose gel for resolution of the DNA fragments by electrophoresis. The 10-kb band corresponding to pCEP4/Kpn1-blunt-ended/HindIII was excised from the gel and purified using the QIAquick® Gel Extraction Kit according to manufacturer’s instructions and eluted in 35 μl of elution buffer. DNA concentration was determined using the NanoDrop Spectrophotometer.

A ligation reaction with the purified insert and receiving vector was carried out with a 1:10 molar ratio using 64 ng of pCEP4 and 96 ng of the PEDF DNA insert in T4 DNA Ligase Reaction Buffer (New England BioLabs) with 400 units of T4 ligase (New England BioLabs) at 16°C for 16 h. A total of 10 μl of the ligation reaction was used to transform 50 μl of One Shot^™ TOP10 Chemically Competent E. coli cells (Invitrogen) according to manufacturer’s instructions. Luria broth (LB) agar plates containing 100 μg/ml of ampicillin were prepared using imMedia^™ Growth Medium, agar, ampicillin (Invitrogen) according to manufacturer’s instructions. A total of 200 μl of transformed cells was plated and spread onto the Luria-Bertani (LB) agar plates, which were inverted and incubated at 37°C for 16 h.

To test for successful ligation, single colonies were picked and inoculated in 50 ml of LB supplemented with 100 μg/ml of ampicillin and incubated at 37°C overnight. The cells were harvested by centrifugation, and the plasmid DNA was purified using the Plasmid Midi Kit (Qiagen) according to manufacturer’s instructions. The nucleotide sequence alterations were confirmed by DNA sequencing of the purified plasmid DNA (Lofstrand Labs).

2.2. Generation of stably transfected HEK.Ebna cells harboring expression vectors for PEDF mutated versions

HEK.Ebna cells (ATCC Cat. # CRL-10852, 293 c18) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin-streptomycin (Gibco) and 0.25 mg/ml Geneticin^™ (Gibco) in a 5% CO₂ humidified incubator at 37°C. For transfection, 3 × 10⁵ cells were plated in one well of a 6-well plate for 16 h. The next day, purified DNA (3.5 μg) of each expression vector in 175 μl of Optimem (Gibco) was mixed with 175 μl of Optimem containing 7 μl of Lipofectamine 2000 (Invitrogen) and incubated for 5 min at room temperature. A total of 250 μl of the mixture (containing 2.5 μg DNA and 5 μl of lipofectamine) was added to one well of a 6-well plate. The cells were incubated in a 5% CO₂ humidified incubator at 37°C for 3 days. The media were then replaced with the selection medium containing DMEM with 10% FBS, 1% penicillin-streptomycin, 0.25 mg/ml Geneticin, and 200 μg/ml hygromycin B (Gibco). The selection medium was added to the untransfected control cells without lipofectamine or optimum and replaced every 2–3 days until all the control cells died, about 8 days.

2.3. Production and purification of mammalian derived PEDF altered proteins

Stably transfected HEK.Ebna cells were cultured in complete medium consisting of DMEM with 10% FBS and 1% penicillin/streptomycin and 0.25 mg/ml Geneticin^™ (Gibco) at 37°C and 5% CO₂. Cells were cultured to confluence in culturing vessels, such as T75 flasks (Corning®) with 75 cm² cell growth area, roller bottles with 850 cm² cell growth area (Corning®), or Corning® CellSTACK® Culture Chambers with 5-Stacks and 3,180 cm² cell growth area, at 37°C. Cells were cycled between serum-containing and serum-free media every 24 h for 5 cycles. The volume of the medium was 50 ml per roller bottle or 650 ml per 5-Stack Culture Chamber. For large scale production, after 2 cycles of serum/no-serum medium in the 5-Stack Culture Chambers, the medium was replaced with FreeStyle^™ 293 Expression Medium (Thermo Fisher) and the cell cultures were incubated before cell dislodging from the surface became evident, usually about 4–5 days. Conditioned serum-free media containing the recombinant proteins were collected and filtered through a 0.45 μm vacuum filter unit. The media from the cycles were pooled and the proteins were precipitated with 80% ammonium sulfate with gentle stirring at 4°C for 16 h. The precipitate was separated by centrifugation, and the pellet was resuspended in PBS pH 7.4 (Gibco) and dialyzed against Buffer S (50 mM Na-phosphate buffer pH 6.4, 50 mM NaCl, 1 mM dithiothreitol (DTT), and 10% glycerol). The dialysate was filtered through membranes of 0.45 micron and used to purify PEDF by cation-exchange column chromatography using a MonoS column pre-equilibrated with 10 column-volumes of Buffer S attached to an automated FPLC system (AKTA). The flow rate was 5 ml per min. The unbound proteins were washed with 20 column-volumes of Buffer S. Bound proteins were eluted with 20 column-volumes of a 50–500 mM NaCl linear gradient in Buffer S. Fractions containing PEDF were identified by immunoblot using specific anti-PEDF antibodies (XpressBio) and then pooled and dialyzed against Buffer Q (50 mM Tris pH 8.0). PEDF was further purified by anion-exchange chromatography using a MonoQ column pre-equilibrated with Buffer Q attached to an automated FPLC system (AKTA). The flow rate was 5 ml per min. The unbound proteins were washed with 20 column-volumes of Buffer Q. Bound proteins were eluted with 20 column-volumes of a 0–1000 mM NaCl linear gradient in Buffer Q. PEDF-containing fractions were identified by immunoblot with anti-PEDF and pooled. PEDF containing fractions were dialyzed against PBS pH 7.4 and stored at −80°C until use. Computation of the theoretical isoelectric point (pI) and molecular weight (Mw) for the PEDF versions were obtained using the Compute pI/Mw tool of Expasy (https://web.expasy.org/compute_pi/) and are shown in Table 1.

Table 1.

Characteristics of PEDF variants

PEDF Variants*	pI	MW	Alterations	Resulting activity	Ref.
PEDF[R99A]	5.84	46227.1	R99A	Loss of binding to PEDF-R	13
PEDF[H105A]	5.9	46246.15	H105A	Enhanced binding to PEDF-R	13
PEDF[Collagen]	6.48	46180.18	D256A, D258A, D300A	Loss of collagen binding	7
PEDF[Heparin]	5.6	46112.91	K146A, K147A, R149A	Loss of heparin binding	8

^*Unaltered PEDF has affinity for PEDF-R, collagen, hyaluronan and heparin, and a pI of 5.97 and MW of 46312.21.

Recombinant human PEDF conjugated with fluorescein (Fl-PEDF) was as described before [28] [18], [26].

2.4. Bacterial strains and plasmids

pEXP1-DEST vectors (Invitrogen) with an N-terminal His₆-Xpress tag under the control of the T7 promoter were the host plasmid for bacterially derived PEDF-R. The Xpress epitope (Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys) comprises the enterokinase (EK) recognition site (Asp-Asp-Asp-Asp-Lys). The coding sequences of the PNPLA2 variants subcloned into pEXP1-DEST vectors as previously described [18], [26] were used. Expression plasmids were propagated in TOP10 or DH5α E. coli cells. BL21(DE3) pLysS One Shot chemically competent E. coli cells (Invitrogen/Thermo Fisher Scientific) were used as the host.

2.5. Expression of bacterially derived PEDF-R polypeptide variants

Ten nanograms of each expression plasmid were used to transform BL21(DE3) pLysS One Shot chemically competent E. coli cells (Invitrogen/Thermo Fisher Scientific) according to manufacturer’s instructions. A total of 200 μl of the transformation mixture was plated and spread onto LB agar plates supplemented with 100 μg/ml of ampicillin, inverted, and incubated at 37°C for 16 h. A single colony was picked from the plate to inoculate a starter culture of 2 ml of LB broth supplemented with 100 μg/ml ampicillin and incubated at 37°C with shaking at 230 RPM for 16 h. Starter cultures were diluted 1:10 in LB supplemented with 100 μg/ml of ampicillin, incubated at 37°C with shaking at 230 RPM. Aliquots of the cultures were used to determine their optical density at 600 nm (OD600) and were monitored until the culture reached an OD600 of 0.6. Recombinant expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) (Sigma), an efficient molecular inducer of transcriptional activity of the T7 promoter [29], and incubation continued at 37°C with shaking for 3 h. Cells were separated from the medium by centrifugation at 2000 × g and stored at −80°C until use. For protein production analyses, cells were resuspended in 200 μl of Buffer P (PBS pH 7.4 and protease inhibitors (cOmplete^™ Protease Inhibitor Tablets, Roche/Millipore Sigma)) per ml of pelleted culture and then disrupted by sonication with a 50% pulse for 20 s on ice using a Sonic Dismembrator (Fisher Scientific, Model 100). Aliquots of 1 ml were centrifuged at 20,800 × g for 10 min at room temperature, and the supernatant was discarded. The pellets were used immediately or stored at −80°C. The particulate material was used to evaluate solubilization in PBS solutions containing 2 M – 8 M urea.

2.6. Purification of bacterially derived PEDF-R polypeptides

For cation-exchange column chromatography of PEDF-R, cell pellets from cultures, as described above, were resuspended in 8 M urea in PBS pH 7.4 with 1 mM DTT, and the suspension was sonicated for 20 s with a 50% pulse, as above. The sonicated cell suspensions were centrifuged at 20,800 × g for 10 min at room temperature and the supernatant was collected. The supernatant was diluted 1:10 in cold Buffer S and applied to a Poly-Prep® Chromatography Column (BioRad) containing a 0.5 ml bed volume of SP Sepharose (GE Healthcare Life Sciences) pre-equilibrated with cold Buffer S with gravity flow. The flowthroughs were collected, and the unbound proteins were washed with 10-column volumes of cold Buffer S. Bound proteins were eluted from the resin using a stepwise gradient of NaCl diluted in cold Buffer S in 1 ml fractions. The samples were collected and stored at −20°C until use. Computation of the theoretical isoelectric point (pI) and molecular weight (Mw) for the PEDF-R versions were obtained using the Compute pI/Mw tool of Expasy (https://web.expasy.org/compute_pi/) and are shown in Table 3.

Table 3.

Characteristics of PEDF-R variants

His6/Xpress-PEDF-R variants	PEDF-R amino acid residues	MW	pI	Resulting activity	Ref.
PEDF-R	M1-L504	63711.66	5.66	PEDF affinity/active enzyme	18; 26
PEDF-RΔ203–232	M1-L504ΔH203-L232	60161.54	5.49	Loss of PEDF binding	26
PEDF-R(D166A)	M1-L504[D166A]	63667.65	5.72	Enzymatically inactive	26; 38
PEDF-R4	M1-L232	31749.94	6.26	PEDF affinity/ active enzyme	26
PEDF-R4Δ203–232	M1-I202	28199.81	5.98	Loss of PEDF binding	26

Alternatively, PEDF-R variants were purified by cation-exchange column chromatography of PEDF-R proteins in a batch fashion. Cells from cultures were separated from medium by centrifugation at 2000 × g 20 min at 4°C, and resuspended with cold Buffer TEN (10 mM Tris-CL pH 7.5, 1 mM Ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl) (100 μl per ml of starting culture) by pipetting up and down and vortexing gently. The suspension was sonicated with 5 bursts of 15s on and 15s off while kept in an ice bath. The particulate material in the sonicated sample was separated from the soluble material by centrifugation at 20,800 × g in an Eppendorf centrifuge for 15 min at 4°C. The pellet was resuspended in PBS pH 7.4 containing 8 M urea at 100 μl per ml of starting culture, vortexed and sonicated with 3 bursts of 15s on and 15s while kept in an ice bath. The suspension was kept on ice for 15 min and then centrifuged at 20,800 × g in Eppendorf centrifuge for 15 min at 4°C. The supernatant contained soluble proteins in PBS pH 7.4 with 8 M urea. The supernatant was diluted at 1:10 (v:v) with Buffer S containing 8 M urea and 50 mM NaCl and applied to Macro Spin Columns (The Nest Group, Inc.) containing 200 μl of pre-equilibrated cation-exchange resin with Buffer S containing 8 M urea and 50 mM NaCl. The flow-through was collected by centrifugation at 1000 × g for 1 min. The resins were washed with 2 ml of Buffer S/8 M urea/50 mM NaCl and bound proteins were eluted with 200 μl of Buffer S containing 8 M urea and 1 M NaCl. The samples were collected and stored at −20°C until use.

2.7. SDS-PAGE and Western blot

Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The samples were mixed with 4X LDS sample buffer (Novex) to achieve a final concentration of 1X LDS sample buffer containing 1.25 mM DTT, followed by heating at 95°C for 5 min. The samples were loaded onto NuPAGE Novex Bis-Tris protein gels (Invitrogen). Proteins were separated in the gel by electrophoresis at constant voltage of 170 Volts for 1 h with NuPAGE MOPS SDS Running Buffer (Invitrogen). Proteins in the gels were stained using the Colloidal Blue Staining Kit (Invitrogen) according to manufacturer’s instructions.

For western blots, proteins resolved by SDS-PAGE were transferred from the gels to nitrocellulose membranes using the iBlot2 Western Blotting System (Invitrogen). For PEDF immunodetection, membranes were incubated in blocking buffer composed of 1% bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBS-T composed of 50 mM Tris-Cl pH 7.5, 150 mM NaCl, and 0.05% Tween 20) at room temperature with gentle rocking for 1 h. Then, the membranes were incubated in a solution of primary antibody against human PEDF (BioProducts MD LLC/XpressBio) diluted 1:100,000 in BSA/TBS-T at 4°C with gentle rocking for at least 16 h. The membranes were washed with TBS-T at room temperature with vigorous rocking for 30 min, and then incubated in a solution of horse radish peroxidase (HRP)-conjugated goat anti-rabbit (Kindlebio) diluted 1:1000 in 1% BSA/TBS-T at room temperature with gentle rocking for 30 min. After washing the membrane as above, the blot was incubated in the Ultra Digital-ECL substrate solution (Kindlebio), a solution with an HRP substrate that emits chemiluminescence and developed with the KwikQuant Imager (Kindlebio).

For PEDF-R immunodetection, the membranes were incubated in blocking buffer composed of 1% BSA in TBS-T at room temperature with gentle rocking for 1 h. Then, the membranes were probed with a primary antibody against human PEDF-R (Sigma) diluted 1:250 in 1% BSA/TBST or Xpress (Invitrogen) diluted 1:100,000 in 1% BSA/TBS-T at 4°C with gentle rocking for at least 16 h. The membranes were washed at room temperature with TBS-T three times for 5 min each with vigorous rocking. Then, the membranes were probed with HRP-conjugated goat anti-rabbit (KPL) diluted 1:10,000 (PEDF-R) or diluted 1:200,000 (Xpress) in 1% BSA/TBS-T with incubations at room temperature with gentle rocking for 30 min. After washing the membrane as above, the blot was incubated in the Pierce^™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and exposed to X-ray films.

2.8. His-Tag pulldown assay

Binding of PEDF to PEDF-R was assayed by His-Tag pulldown of protein complexes using Ni-NTA resin. Solubilized E. coli inclusion bodies containing PEDF-R were refolded by rapid dilution and concentrated with Amicon® Ultra-4 Centrifugal Filter Units with 10000-MWCO for buffer exchange in binding buffer (50 mM Tris pH 7.5, 100 mM NaCl, 3 mM deoxycholate). Then, 1 μg of total protein (determined by measuring absorbance at OD 280nm using NanoDrop) was mixed with 200 ng of Fl-PEDF in binding buffer to a total volume of 50 μl and incubated on ice for 1 h to allow complex formation. Given that PEDF-R was ∼80% of total protein, we estimated that the amount of PEDF-R in each reaction was ∼800 ng. A total of 25 μl of Ni-NTA beads (Thermo Fisher Scientific) were washed and equilibrated with the binding buffer. The protein Ni-NTA beads were mixed and incubated on ice for 1 h with gentle agitation. The beads were sedimented by centrifugation at 800 × g at 4°C for 5 min. The unbound material in the supernatant was collected. A total of 50 μl of load and unbound material was added to a well of Corning 96-Well Clear Bottom Black Polystyrene Microplates (Corning). Fluorescence of Fl-PEDF was measured using Spectramax M5 (Molecular Devices) at excitation 485 nm and emission 510 nm. The percentage of bound protein was determined by subtracting the unbound from the total (load).

3. Results

3.1. Mammalian overexpression of PEDF variants

The characteristics of the PEDF variants with altered biological activities used in this study are summarized in Table 1. Construction of the expression vectors for the PEDF[H105A], PEDF[R99A], PEDF[Collagen] and PEDF [Heparin] variants were described in methods and illustrated in Figure S1. We transfected HEK.Ebna cells with the expression plasmids and selected the ones resistant to hygromycin B. Given that PEDF is a secreted protein, we tested the culturing media of small cultures of the stably transfected cells for production of the recombinant proteins by western blots. The serum-free medium of transfected cells contained the recombinant proteins, and PEDF was not detected in medium from untransfected HEK.Ebna cells (Fig. 1A). Then, cultures were scaled up and the cells were incubated in medium alternating with and without serum for about 24 h each for a total of 5 cycles. The recombinant protein production in the harvesting media (without serum) increased with each cycle, as detected by western blotting (Fig. 1B). We estimated that the recombinant PEDF produced by the cells amounted to ~90% of the total protein of the conditioned serum-free media of cycle 6 as from Coomassie blue staining of proteins after SDS-PAGE (Fig. 1C). The results demonstrate that the stably transfected HEK.Ebna cells produced and secreted the recombinant proteins into the medium, indicating that the plasmids are functional expression vectors for the PEDF variants.

Figure 1. Expression of PEDF variants in HEK.Ebna cells.

Stably transfected and selected HEK.Ebna cells were cultured in complete medium. The medium was removed, and the cells were incubated with serum-free medium for the indicated times. (A) Western blot of untransfected and stably transfected HEK.Ebna cells incubated with serum-free medium for 24 hours. (B) Stably transfected cells were cycled between serum and serum-free medium for a total of 5 cycles, and 20 μl of conditioned medium from each serum-free medium cycle was analyzed by western blot versus ab-PEDF. (C) A total of 100 μl of conditioned serum-free media from cycle 6 was concentrated by Amicon ultra-4 centrifugal filters, resolved by SDS-PAGE, and analyzed by Coomassie blue staining.

3.2. Purification of recombinant PEDF protein variants

We used the serum-free media conditioned by stably transfected HEK.Ebna cells with the human PEDF expression vectors as starting material to produce and purify the recombinant proteins. The purification protocol included protein concentration by 80% ammonium sulfate precipitation followed by a two-step ion exchange column chromatography. Proteins in the concentrated media were subjected to fractionation by cation-exchange column chromatography with a column of MonoBeads S strong cation-exchange chromatography resin. Figure 2 shows chromatograms of each PEDF variant. PEDF[Heparin] did not bind to the cation-exchange resin. Wild type PEDF, PEDF[R99A], and PEDF[H105A] eluted with 250 mM NaCl, while PEDF[Collagen] eluted with 300 mM NaCl. The PEDF[H105A] variant was further purified by anion-exchange column chromatography. The fractions containing PEDF[H105A] were pooled, dialyzed against Buffer Q and the protein was subjected to anion-exchange column chromatography. The fractions collected from the chromatography were resolved by SDS-PAGE and analyzed by Coomassie blue staining and western blotting (Figs. 3A and 3B). Fractions containing PEDF[H105A] were pooled, concentrated, and resolved by SDS-PAGE for protein staining with Coomassie blue and immunostaining with a specific anti-PEDF antibody. Figure 3C shows that PEDF[H105A] was obtained in the final fraction with high purity. The yields of PEDF[H105A] are summarized in Table 2. From the estimated starting material of 25 mg/liter of PEDF[H105A] at about 40–50% purity, the estimated yield of purified PEDF[H105A] was ~1mg/liter at about 99% purity.

Figure 2. Cation-exchange chromatography of PEDF variants.

Representative chromatograms of the cation-exchange column chromatography from each PEDF variant. Conditioned serum-free medium cycles from stably transfected HEK.Ebna cells were pooled, concentrated by ammonium sulfate precipitation, and dialyzed against Buffer S. Dialysate was loaded onto a Mono S column pre-equilibrated with Buffer S. After protein loading, a 10-column volume (CV) wash was performed to further elute unbound proteins. Bound proteins were then eluted by a 20 CV NaCl gradient from 50–500 mM, and the peaks were collected.

Figure 3. Anion-exchange column chromatography of PEDF[H105A].

(A) Fractions collected from cation-exchange chromatography were pooled and dialyzed against Buffer Q. Dialysate was loaded onto a Mono Q column pre-equilibrated with Buffer Q. After protein loading, a 10 CV wash was performed to further elute unbound proteins. Bound proteins were then eluted by a 20 CV NaCl gradient from 0 to 1000 mM, and the peaks were collected. Fractions were analyzed by Coomassie blue staining. (B) Western blot of fractions as in panel A against antibody to PEDF. (C) Pooled fractions from anion-exchange chromatography were concentrated using Amicon ultra centrifugal filters and were resolved by SDS-PAGE. Representative Coomassie blue staining (CB) and western blot of purified PEDF[H105A].

Table 2.

PEDF[H105A] purification table

Sample	Concentration (mg/ml)	Estimated purity (%)
Media	0.025	40–50
Ammonium Sulfate precipitation + Dialysis	6.7	50–60
S-Sepharose	1.9	95
Q-Sepharose	0.48–1	99

3.3. Bacterial overexpression of PEDF-R variants

The expression vectors of human PEDF-R variants in pEXP1-DEST vectors contain an N-terminal His₆-Xpress tag [18], [26]. Figure 4A shows the organization of the plasmid and Fig. 4B shows the amino acid residues of each of the PEDF-R variants, expected molecular weight based on the length of their open reading frames, and biological activities. The full-length wild-type PEDF-R protein binds to PEDF and has lipase activities. The PEDF-RΔ protein lacks the binding site for PEDF. The PEDF[D166A] variant contains a single amino acid substitution at aspartic acid 166, which forms part of the catalytic dyad and therefore a replacement to alanine renders the protein enzymatically inactive. The PEDF-R4 variant is derived from the first four coding exons of the PNPLA2 gene and retains enzymatic activity and binds PEDF. Finally, the PEDF-R4Δ variant is a truncated form of PEDF-R4, which lacks the PEDF binding site but remains enzymatically active.

Figure 4. Construction scheme of PNPLA2 variants.

(A) PNPLA2-containing expression vectors for PEDF-R variants were constructed into pEXP1-DEST expression vectors with an N-terminal His₆-Xpress tag under the T7 promoter as previously described [18], [26]. (B) Table summarizing the PEDF variants, amino acid residues, expected molecular masses, and resulting activities. Sequence schematic of the PEDF-R variants is given to the right of the table.

E. coli cells were transformed with the expression vectors harboring the cDNA for each PEDF-R variant. The cells were grown to an optical density of 600 nm, induced with IPTG for 3 h, and harvested. The cells were lysed, and the soluble and insoluble fractions of the lysed cells were separated and resolved by SDS-PAGE and western blots. The PEDF-R proteins were present in the particulate material (Fig. 5A). The main bands in SDS-PAGE gels stained with Coomassie Blue corresponded to proteins that migrated with the expected apparent molecular weight for each PEDF-R variant (compare migration patterns in Fig. 5A with theoretical MW in the Table 3). Western blotting against anti-Xpress (Fig. 5B) and anti-PEDF-R (Fig. 5C) confirmed that those bands were of proteins of expected N-Xpress tagged-PEDF-R proteins. The yields of the recombinant proteins estimated from Coomassie blue staining of gels as in Fig. 5A were of several milligrams for the different PEDF-R proteins per liter of culture (Fig. 5D). Given that the proteins were insoluble in aqueous solutions, they were challenged with urea for solubilization. For this purpose, the particulate material was incubated in solutions containing increasing concentrations of urea. Ponceau red staining and western blotting of the soluble and insoluble fractions showed that recombinant PEDF-R variants were partially solubilized in 4M urea, with increasing solubility in 6M urea and 8 M urea (Fig. 6). Note that the main band stained with Ponceau Red is the one for the recombinant proteins demonstrating high purity of the proteins in the fractions (estimated ~70%). The results demonstrate that bacterially derived PEDF-R proteins were produced in large amounts in insoluble inclusion bodies that can be solubilized with urea.

Figure 5. Bacterially derived recombinant PEDF-R variants expressed in E. coli.

BL21 E. coli cells were transformed with PNPLA2-containing pEXP1-DEST expression vectors and streaked onto agar plates containing ampicillin. A single colony was used to generate an overnight culture, which was diluted 1:10 in LB, grown to an optical density of 0.6 at 600 nm, and induced with 1 mM IPTG for 3 h. Cells were pelleted by centrifugation and lysed by sonication in Buffer P. Soluble (S) and particulate material (P) from bacterial extracts obtained after cell lysis were analyzed by SDS-PAGE and western blot for protein expression. (A) Coomassie blue-stained gel indicating the correct apparent molecular mass for the N-terminal His₆/Xpress-tagged PEDF-R polypeptides produced in E. coli. Western blot of soluble and insoluble bacterial extracts versus (B) anti-Xpress and (C) anti-PEDF-R. (D) Protein yields of PEDF-R variants. PEDF-R (R); PEDF-RΔ (RΔ); PEDF-R4 (R4); PEDF-R4Δ (R4Δ).

Figure 6. Solubilization of recombinant variants with urea.

Particulate material from E. coli cell lysates was resuspended in solutions containing increasing concentrations of urea. The soluble and insoluble fractions were separated by centrifugation. Samples were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Ponceau red staining (top) and western blot versus Ab-Xpress (bottom).

3.4. Purification of PEDF-R variants

The PEDF-R variants were subjected to cation exchange chromatography. To purify PEDF-R, we diluted the urea-solubilized cell lysates in Buffer S and applied the filtrate to a pre-equilibrated cation-exchange resin. The bound proteins were eluted using a stepwise NaCl gradient, and full-length PEDF-R eluted with 200 mM NaCl. Coomassie blue staining after SDS-PAGE showed that PEDF-R was ~90% pure (Fig. 7A). PEDF-R4 and PEDF-R4Δ subjected to cation-exchange column chromatography also bound and eluted from the resin with 1M NaCl (Fig. 7B). The results demonstrate that PEDF-R variants can be purified by subjecting them to cation-exchange chromatography.

Figure 7. Cation exchange of recombinant PEDF-R variants.

(A) Particulate material from cell lysates from E. coli cells overexpressing recombinant full length PEDF-R was solubilized with 8 M urea and then purified by stepwise elution cation-exchange chromatography. The fractions were resolved by SDS-PAGE and analyzed by Coomassie blue staining. I – Input; FT – flowthrough (B) Particulate material from cell lysates from E. coli cells overexpressing recombinant PEDF-R4 and PEDF-R4△ was diluted in cation-exchange chromatography buffer containing 8 M urea was applied to a cation-exchange column and eluted (E) with 1M NaCl. Fractions were resolved by SDS-Page and analyzed by Coomassie blue staining.

3.5. Binding of Fl-PEDF to PEDF-R variants

To determine the binding of PEDF to the PEDF-R variants, Fl-PEDF was mixed with the variants that contain a His-tag fusion region and His-tag pulldown assays were performed. The observed variability of the assay is likely due to loss of Ni-NTA beads during washings among experiments, binding affinity differences among Ni-NTA beads batches to the His-Tag, and/or uncontrolled Fl-PEDF precipitation during the assay. PEDF-R, PEDF-R[D166A], and PEDF-R4 exhibited PEDF binding, while PEDF-RΔ203–232 and PEDF-R4Δ203–232 did not, (Fig. 8) indicating that the recombinant PEDF-R proteins behaved as expected [26].

Figure 8. Binding of Fl-PEDF to recombinant PEDF-R variants.

A total of 200 ng of Fl-PEDF was incubated with 1 μg of total protein from cell lysates overexpressing each PEDF-R variant. Protein complexes were subjected to a His-tag pulldown in binding buffer containing 50 mM Tris pH 7.5, 100 mM NaCl, and 3 mM deoxycholate. The unbound material was collected, and the fluorescence of the load (L) and unbound material (UB) was measured by the Spectramax plate reader. % Bound = L-UB/L*100. The results from two separate experiments for Fl-PEDF binding to PEDF-R, labeled as PEDF-R1 and PEDF-R2, and to PEDF-R4, labeled as PEDF-R41 and PEDF-R42, are shown. Supplementary Information

4. Discussion

In this study, we have shown that the mammalian HEK.Ebna and bacterial E. coli cells are viable and economical production systems for recombinant human PEDF and PEDF-R variants. They are efficient and produce significant amounts of each of these proteins at a high percentage of the total protein. The recombinant PEDF proteins are produced in milligram amounts per liter of cultures of HEK.Ebna cells stably transfected with the pCEP4-based expression vectors harboring cDNA of multiple PEDF variants. The apparent molecular weight of PEDF[R99A], PEDF[H105A], PEDF[Collagen] and PEDF[Heparin] matches that of the wild type PEDF (50,000-MW). The PEDF variants, except for PEDF[Heparin], can be purified by cation-exchange chromatography. Due to the reduced availability of positively charged amino acids in the PEDF[Heparin] variant, the protein lacks affinity for the negatively charged resin. The remaining PEDF variants can be purified using the two-step chromatography protocol optimized in this study from the harvesting media.

Comparison of our HEK.Ebna expression system for PEDF with mammalian expression systems reported previously present several differences and advantages. 1) Epitope tags are often used to improve purification strategies; however, they may affect the biological functions of proteins. Previously, we produced recombinant PEDF, PEDF[H105A] and PEDF[R99A] in baby hamster kidney (BHK) cells using the p3xFLAG-CMV-9 expression vector containing PEDF cDNA with an N-terminal 3xFLAG tag [13]. While the proteins displayed expected biological functions, the neurotrophic effects of PEDF and PEDF[H105A] were lower than those observed in experiments with untagged proteins [13]. An advantage of the expression system presented here for PEDF using the pCEP4 expression vector in HEK.Ebna cells is that the protein is expressed free of epitope tags, which could interfere with biological assays. 2) The complete medium for cultured HEK.Ebna cells includes both G418 and penicillin/streptomycin, providing added protection against contamination from unwanted cells. As transfected HEK.Ebna cells are resistant to G418, this neomycin analog antibiotic cannot be used to select stably transfected cells, and therefore hygromycin B was used for the selection of transfected cells. Hygromycin B is an aminoglycoside antibiotic that kills prokaryotic and eukaryotic cells by inhibiting protein synthesis [30]. The addition of hygromycin to the medium of stably transfected cells provides additional protection against contamination. 3) The estimated yield of PEDF[H105A] and other altered PEDF variants from pCEP4-PEDF transfected HEK.Ebna cells is in the milligram range per liter of culture, which is similar to the levels of production of PEDF, antithrombin III and alpha 1-proteinase inhibitor, which are other members of the SERPIN superfamily, from stably transfected BHK cells with respective expression vectors[31] [32], [33]. For the BHK cell expression system, co-transfection of the pBK-RSV and pMaStop expression vectors containing the coding sequences under the control of the Rous sarcoma virus (RSV) and simian virus 40 (SV40) promoters, respectively, along with the pMAStop, pSV2dhfr, and pRMH140 plasmids is performed, and the cells are cultured with methotrexate and neomycin to maintain selection. In contrast, our pCEP4 expression vector contains PEDF coding sequences under the control of the cytomegalovirus CMV promoter and selection of cells containing the expression vector is achieved by resistance to hygromycin B. Thus, the protocol presented in this study details a method to produce recombinant PEDF variants by transfecting and selecting HEK.Ebna cells using a single plasmid to produce PEDF proteins free of epitope tags in high yields. 5) The expression system presented here is for recombinant PEDF variants that are produced as soluble, folded, and glycosylated proteins in contrast with production by bacterial expression systems. Previously, a bacterial system with expression plasmids containing coding sequences for human PEDF full-length and N-terminus truncated PEDF (44–418 amino acids), yielded undetectable full-length protein, while the truncated PEDF was produced forming inclusion bodies and lacked glycosylation [34]. The truncated PEDF was insoluble in aqueous solutions and required 4M urea to be brought into solution. Thus, production of PEDF variants in the mammalian expression system with HEK.Ebna cells, as described here, presents an advantage over the bacterial expression system and it yields PEDF proteins with native suitability for biological and structural studies.

In this study, we have shown that recombinant PEDF-R variants can be produced in BL21 E. coli cells. Comparison of the expression system for PEDF-R described here with those previously reported reveals several advantages. In other studies, active PEDF-R has been expressed in systems using COS-7, HEK293 and SF9 cells [21], [34], [35]. The production of purified protein in those systems was lower than the one described here for recombinant PEDF-R proteins in bacteria. The yields of PEDF-R variants using BL21 E. coli cells are higher than our previous expression system in which PEDF-R was synthesized by E. coli lysates using a commercial cell-free in vitro protein translation system [26]. In our system, E. coli cell pellets or lysates can be readily stored at −80°C, and then thawed, solubilized with urea, and used for experiments the same day, which allows for the possibility of running multiple experiments in a timely fashion. The rapid dilution and buffer exchange steps for solubilizing the recombinant PEDF-R proteins is an approach preferred over other methods, such as dialysis or slow dilution in buffers with detergents, etc., which require longer time to achieve protein solubilization and refolding.

We have also demonstrated that PEDF-R can be highly purified from cation-exchange chromatography. The PEDF-R variants produced in this study possess an N-terminal His₆-Xpress tag that can be also exploited for purification and for performing binding experiments. An added advantage is that this epitope tag can be removed if the tag is interfering with the biological functions of the proteins, given that the fused peptide has an TEV protease site for cleavage. We note that the PEDF-R proteins were >70% of the total protein in the particulate material from the cell host under the conditions described. His-tagged PEDF-R variants with the PEDF binding site retained the ability to bind PEDF, while PEDF-R variants lacking the binding site did not, indicating that the epitope tag does not negatively affect native protein activity. Previous reports demonstrate that PEDF-R proteins containing epitope tags retain binding and lipase activities [20], [26], [35], [37], [38].

In conclusion, we obtained large amounts of recombinant proteins of the serpin PEDF and its receptor PEDF-R from recombinant expression systems. Highly purified proteins will prove useful for use in the investigation of the nature of the interaction between PEDF and PEDF-R and structural biology.

Supplementary Material

1

Figure S1. Construction scheme for pCEP4 plasmid. PEDF cDNA was excised from the pBk-CMV expression vector by digestion with HindIII and EcoRI. The PEDF coding sequence was inserted into the pCEP4 vector between KpnI and HindIII restriction sites under the control of the CMV promoter.

Highlights.

• Expression vectors for human PEDF protein mutated versions were constructed.

• Recombinant human PEDF protein versions were produced and purified from mammalian cell cultures in milligram amounts.

• Recombinant human PEDF-R protein truncated versions were produced and purified from E. coli cells in milligram amounts.

• Fluorescein-conjugated PEDF bound to recombinant PEDF-R versions.

Abbreviations:

PEDF

Pigment epithelium-derived factor

PEDF-R

PEDF receptor

E. coli

Escherichia coli

SERPIN

serine protease inhibitor

RPE

retinal pigment epithelial

DHA

docosahexaenoic acid

FBS

fetal bovine serum

DMEM

Dulbecco’s modified Eagle’s medium

PBS

phosphate buffered saline

DTT

dithiothreitol

FPLC

Fast protein liquid chromatography

IPTG

isopropyl-β-d-thiogalactopyranoside

OD

optical density

EDTA

ethylenediaminetetraacetic acid

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

BSA

bovine serum albumin

HRP

horse radish peroxidase

NTA

nitrilotriacetic acid

CMV

cytomegalovirus

RSV

Rous sarcoma virus

SV40

simian virus 40