Production and Purification of an Optimized Osteopontin Peptide for Use in Vascular Calcification Treatment Studies

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1 Production and Purification of an Optimized Osteopontin Peptide for Use in Vascular Calcification Treatment Studies Erin Martin, 1 Ashwini S. Pai, 2 Mandy Lund, 2 Cecilia M. Giachelli 2 1 Department of Biology, Western Washington University, Bellingham, Washington Department of Bioengineering, University of Washington, Seattle Washington Abstract: We have previously shown that native, full length murine osteopontin (OPN), an anti-calcific protein, was able to prevent vascular calcification (VC) in a uremic mouse model. An optimized murine bioactive and bioengineered N-terminal OPN peptide (mnopn), is being investigated as a preventative and as a treatment to reverse VC. The goal of the present study was to produce 5 mg of purified, recombinant mnopn by subcloning mnopn cdna into a pqe-30 vector, transforming SURE 2 E. coli, and purifying by nickel-nitrilotriacetic acid (Ni- NTA) column method. Identity was confirmed by SDS-PAGE and western blots using anti-opn and anti-his primary antibodies. The initial purification process yielded a non-optimal product. This process was further refined and a time-course was conducted. A three hour growing period after IPTG induction was determined to be optimal. In future studies, the purified recombinant bacterial mnopn will be used in preclinical mouse studies of VC. 1. INTRODUCTION Cardiovascular disease affects over 81 million people in the United States [1] and it is the leading cause of death for sufferers of chronic kidney disease (CKD) [6]. Vascular calcification (VC) is a major indicator of mortality from heart disease, and those with end stage kidney disease are particularly at risk due to improper excretion of minerals [3,4]. High levels of phosphate have been shown to induce calcification in populations of vascular smooth muscle cells [3,4]. When the aorta becomes calcified, the heart must work harder to pump blood to the rest of the body, leading to left ventricular hypertrophy and hypertension, both of which contribute to heart failure and increased mortality [3]. Osteopontin (OPN) is a naturally occurring anti-calcific protein that regulates mineralization by binding to minerals, thereby preventing crystal formation [4,5]. Additionally, it signals osteoclasts to acidify the environment to a level that dissolves minerals [4]. Osteopontin has been shown to prevent calcification [4]. The arginine-glycine-aspartate (RGD) sequence of amino acids is thought to be important for the inhibitory action of OPN [4]. It is of interest whether a highly optimized OPN fragment may be a successful treatment to reverse pre-existing vascular calcification. The goal of the study was to produce 5 mg of highly optimized bioactive murine N-terminal OPN peptide (mnopn) to be used in a murine VC regression study. 2. MATERIALS AND METHODS 2.1 Subcloning and Transformation The initial phase of the mnopn production involved subcloning mnopn cdna into the Qiagen pqe-30 vector between the BamHI and KpnI cloning sites (Figure 1). The mnopn is an N-terminal peptide that mimics a protease cleaved fragment. The vector attached an arginine-glycine-serine-(histidine) 6 (RGS-His or 6x-His) sequence to the insert, which was utilized for the subsequent purification of the expressed protein. Following subcloning of the mnopn into the vector, SURE-2 supercompetent E. coli were transformed and transformed cells were selected for on plates containing Ampicillin. 2.2 Time-Course Analysis of mnopn Expression A time course was conducted to determine the optimal growing period after isopropyl β-d-1 thiogalactopyranoside (IPTG) induction for maximal mnopn expression and minimal degradation. A colony expressing mnopn was used to inoculate 3 ml LB broth containing 3 µl of 100 mg/ml Ampicillin and the culture was grown overnight at 37 C with shaking. The overnight culture was diluted into 100 ml LB/Amp 100 and grown to an optical density at

2 Figure 1. Qiagen pqe-30 vector map displaying the location of the inserted mnopn sequence. 600 nm of 0.7. IPTG was added to a concentration of 1 mm to induce production of the mnopn by inhibiting the repressor of the lac operon. Aliquots were taken prior to induction and at 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h postinduction. Volumes of aliquots were adjusted to normalize for differences in optical densities between sampling times. Samples were centrifuged for 1 min at 15,000 x g and the pellet was resuspended in 100 µl of lysis buffer (89 ml sonication buffer (50 mm NaH 2 PO 4, 30 mm NaCl, 20 mm imidazole, ph 7.8) and 11 ml 10% Triton-X). Cells were lysed by gentle vortexing and sonication until the solution was translucent. Lysate, purified mnopn, and recombinant mouse OPN from R&D systems were run on SDS-PAGE gels for analysis with ISS Pro-Blue staining, Millipore amido black staining, and western blotting. 2.3 Optimized Ni-NTA Purification of 6x-Histagged Protein A colony expressing the mnopn was restreaked for single colonies, which were then used to inoculate 15 ml overnight LB broth cultures containing 15 µl of 100 mg/ml Ampicillin. Cultures were grown overnight at 37 C with shaking, diluted 1:50 in 500 ml LB/Amp 100, and grown until an optical density at 600 nm of 0.7 to 0.9 was reached, at which point production of mnopn was induced by the addition of IPTG to a concentration of 1mM. After growing for a further three hours, cultures were centrifuged at 4000 x g for 10 min at 4 C. The supernatant was removed and the cell pellets were frozen overnight at -20 C. Pellets were resuspended in 20 ml sonication-lysis buffer (178 ml sonication buffer (50mM NaH 2 PO 4, 30mM NaCl, 20mM imidazole, 10% glycerol, ph 7.8) and 22 ml 10% Triton X-100). Protease inhibitors were added (1:5000 of 1 mg/ml Pepstatin, 1:1000 of 100 mm PMSF, 1:1000 of 0.5 mg/ml Leupeptin, and 1:1000 of 2 mg/ml Aprotinin) and cells were sonicated on ice for two 45 sec bursts on low. After the addition of 5 µg/ml Dnase I and 10 µg/ml RnaseA, solutions were centrifuged at 10,000 x g for 20 min at 4 C to pellet cellular debris. Water soluble mnopn remained in the supernatant and was transferred into a beaker with 4 ml of 50% Qiagen Ni-NTA resin that had been equilibrated in sonication buffer. Ni-NTA resin binds His-tagged protein, allowing it to be purified from contaminating proteins (Figure 2).

3 Figure 2. Ni-NTA column purification diagram. High concentrations of imidazole, which has a similar ring structure to histidine, were used to elute the tagged protein from the column. The solution was stirred for 60 min 4 C to allow the 6x-His-tagged protein to bind to the resin. The resin was loaded into a column and washed with 20 ml sonication buffer, 20 ml high salt wash buffer (50 mm NaH 2 PO 4, 900 mm NaCl, 10 mm imidazole, 10% glycerol, ph 6.0), 20 ml wash buffer (50 mm NaH 2 PO 4, 300 mm NaCl, 20 mm imidazole, 10% glycerol, ph 6.0), and finally the 6x-His-tagged protein was eluted from the column with 8 ml elution buffer (50 mm NaH 2 PO 4, 300 mm NaCl, 0.2 M imidazole, 10% glycerol). 1 ml aliquots were collected from each wash step and final eluate was collected in eight 1 ml fractions. The amount of protein present in each fraction was determined by Thermo Scientific Micro BCA Protein Assay. 2.4 SDS-PAGE Analysis and Western Blot Column fractions were analyzed on 1 mm 12.5% acrylamide gels. 5x loading dye with β- mercaptoethanol was added to the samples and the samples were boiled for 5 min at 95 C prior to loading onto the gel. 20 µl samples containing 3 µg of protein were loaded to each lane. Gels were run at approximately 150 V. Proteins on the gel were transferred to a PVDF membrane by electrophoresis at 100 V for 1 h in ice-cold transfer buffer. The membrane was blocked with 3% bovine serum albumin in TBS-T instead of the traditional milk blocking, as milk contains OPN and would therefore interfere with analysis of the purified mnopn. Monoclonal anti-polyhistidine antibody from R&D systems and monoclonal RGS-His antibody from Qiagen, both of which detect the N-terminal 6x-His tag, were used as primary antibodies with horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-mouse antibody from Jackson ImmunoResearch Laboratories used as a secondary antibody. Additionally, anti-opn goat polyclonal OP-199 was used as a primary antibody with HRP rabbit anti-goat secondary antibody. Western Lightning chemiluminescence reagents from PerkinElmer were used for development. 3. RESULTS 3.1 Time Course Analysis Four bands were detected by both anti-opn and anti-his primary antibodies. A fifth band was detected in the western using OP-199 primary antibody, but this would be removed if the lysate were to be column purified, since the 6x-His-tag is not present. The time course analysis indicated that a growing period of three hours after IPTG induction is optimal, due to the peak in production of the band at approximately the same molecular weight (16 kda) as the desired mnopn fragment (Figure 3). This growing period was used in subsequent purifications 3.2 BCA Protein Assay The BCA assay results indicate a total yield of 3 mg highly purified mnopn from 500 ml culture using the optimized purification protocol (Figure 4). The initial un-optimized protocol produced a higher protein yield (8.5 mg from a 500 ml culture), though BCA assay only indicates presence and not purity of proteins. The flowthrough contained the highest concentration of protein, as untagged proteins washed through the

4 Figure 3. Western blot of mnopn production time-course using RGS-His primary antibody and HRP goat antimouse secondary antibody. Lane 1: standard molecular weight ladder, 2: purified His-tagged mnopn, 3: R&D mouse OPN, 4: pre-induction, 5: 30min, 6: 1h, 7: 2h, 8: 3h, 9: 4h, 10. column. Protein concentration then decreased through subsequent washes, until concentration increased again with addition of elution buffer to the column. The greatest concentration of purified mnopn was eluted in the third fraction (E3). 3.3 Western Blots of Purified Eluate Four bands were observed on the Western blots at approximately 30 kda, 20 kda, 15 kda, and 9 kda (Figure 5). The predicted molecular weight of the mnopn with attached RGS-His epitope is approximately 16 kda. All bands were detected by both anti-opn OP-199 and anti-his RGS-His primary antibodies and none of the bands were present in empty vector controls. Optimization of the purification protocol failed to reduce the number of bands obtained. 4. DISCUSSION The total yields obtained were reasonable considering the end goal of producing 5 mg of highly purified mnopn; however, a single peptide of the desired molecular weight was unable to be isolated. Though the initial purification resulted in a higher total yield of protein, it is believed that the optimized protocol produced a more pure product. The third band from the top of the gel was expected to be the desired band, due to its similarity to the predicted size of the recombinant mnopn. However, other studies have shown inconsistencies between predicted recombinant OPN size and inferred molecular size based on SDS-PAGE analysis, with proteins appearing larger than predicted based on sequence [2]. Therefore, size alone is an inadequate identifier, and further analysis is necessary to confirm the identity of the bands. The multiple bands were detected with both anti-opn and anti-his antibodies, indicating that they are all some form of 6x-His-tagged mnopn. Because the same bands are present in the time-course analysis and are visible within 30 minutes after production of the peptide was induced, it is likely that they are not results of degradation during purification. Protease inhibitors were added only after peptide production, so it is possible that the bands resulted from proteolysis during the growing period. The four protease inhibitors used during purification were inhibitors of serine, aspartate, and cystein proteases [7]. No inhibitors of metalloproteinases were included so it is possible that metalloproteinases present in E. coli further degraded the mnopn. Additionally, the larger bands could be dimers, though this is unlikely because no cystein residues are present in the mnopn sequence, so disulfide bonds between peptides would not form.

5 Figure 4. BCA protein assay results from optimized Ni-NTA purification of 6x-His tagged mnopn. Figure 5. Western blot of Ni-NTA column purification elution fractions using an optimized protocol and three hour growing period post-induction. RGS-His primary antibody and HRP goat anti-mouse secondary antibody. Developed with Western Lightning chemiluminescence reagents using a 30 sec exposure time. Lane 1: ladder, 2: flow-through, 3: wash 1, 4: wash 2, 5: wash 3, 6: elution fraction 2 (E2), 7: E3, 8: E4, 9: E5, 10: previous mnopn purification E2.

6 As the end goal of the project is to produce a protein drug for treatment of VC, isolation of a single product is important. If an impure product was used, there would be uncertainties as to the exact cause of the treatment study results, and additionally it could invoke an immune response. For example, if the larger bands are in fact protein aggregates, we would want to remove these prior to using the mnopn in treatment studies, as protein aggregates have been shown to be a factor contributing to the immunogenicity of protein therapeutics [8]. In addition to impurities being potentially immunogenic, they may also increase the immunogenicity of the protein drug, thereby rendering it less effective as a treatment [8]. Once the correct band is identified, by methods such as mass spectrometry, subsequent purification by alternate methods could isolate it from the remaining three bands. 5. Jahnen-Dechent W, Schafer C, Ketteler M, McKee M. Mineral chaperones: a role for fetuin-a and osteopontin in the inhibition and regression of pathologic calcification. J Mol Med. 2008; 86: National Kidney Foundation. August dex.cfm. 7. Roche Applied Science. April Swanson, S. Immunogenicity issues in the development of therapeutic proteins. International Journal of Pharmaceutical Medicine. 2007; 21(3): ACKNOWLEDGEMENTS This work was funded by the National Science Foundation (NSF ) and the Life Sciences Discovery Fund (LSDF) # Additional thanks to Dr. Eric Chudler and Janet Wilt for directing the UWEB REU program and to Drs. Ceci Giachelli, Ashwini Pai, Meiting Wu, and Hsueh Yang for their guidance and support. REFERENCES 1. American Heart Association. January ml?identifier= Gao Y, Agnihotri R, Vary C, and Liaw L. Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments. Matrix Biology. 2004; 23: Giachelli C. The emerging role of phosphate in vascular calcification. Kidney International. 2009; 75: Giachelli C, Speer M, Li X, Rajachar R, Yang H. Regulation of vascular calcification: roles of phosphate and osteopontin. Circulation Research. 2005; 96: