- Email: [email protected]
Production and purification of recombinant human SPARC
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19
Production and purification of recombinant human SPARC
Gail Workman*, Amy D. Bradshaw†,‡,1 *Matrix Biology Program, Benaroya Research Institute, Seattle, WA, United States Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, SC, United States ‡ Ralph H. Johnson Department of Veteran’s Affairs Medical Center, Charleston, SC, United States 1 Corresponding author: e-mail address: [email protected] †
CHAPTER OUTLINE 1 Introduction ........................................................................................................336 2 Method...............................................................................................................337 2.1 Background ........................................................................................337 2.2 Insect Cell Culture...............................................................................337 2.3 Baculoviral Expression of Human SPARC ..............................................338 2.4 Maintenance of Viral Vector..................................................................339 3 Purification of Recombinant SPARC Produced by Insect Cells ................................339 3.1 Background ........................................................................................339 3.2 Experimental Method...........................................................................340 3.3 Analysis and Storage of Recombinant SPARC ........................................341 References ............................................................................................................. 343
Abstract The matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or as BM-40) is a collagen-binding protein with a capacity to induce cell rounding and influence proliferation in cultured cells. In mice that do not express SPARC, fibrillar collagen is reduced in some adult tissues; notably, a reduction in fibrosis is reported in response to fibrotic stimuli in lungs, heart, skin, liver, and in the eye. Recently, mutations in the gene encoding SPARC were found in patients afflicted with osteogenesis imperfecta. Thus, SPARC appears to be a critical mediator of collagen deposition and assembly in tissues. A useful tool for assessing the function of SPARC in ECM assembly is a source of purified recombinant SPARC. Outlined in this chapter is a brief discussion of different strategies for generating recombinant SPARC and an experimental strategy for producing and purifying human recombinant SPARC driven by baculoviral expression in insect cells. Methods in Cell Biology, Volume 143, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2017.08.020 © 2018 Elsevier Inc. All rights reserved.
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1 INTRODUCTION SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein involved in tissue remodeling and fibrosis (Bradshaw, 2012; Trombetta-Esilva & Bradshaw, 2012). Mutations in the gene encoding SPARC were recently associated with a type of osteogenesis imperfecta, a finding that reinforces the hypothesis that SPARC is critical for collagen deposition in mineralized tissues as well as in nonmineralized tissues (Mendoza-Londono et al., 2015; Rosset & Bradshaw, 2016). Typical of most matricellular proteins, SPARC has a modular structure. SPARC has three domains (Brekken & Sage, 2001). The N-terminal domain contains a high-capacity, low-affinity Ca2+-binding site, with a sequence located in the first 10 amino acids that serves as a transglutaminase recognition site. The second domain is characterized by a follistatin-like domain with similarities to the Kazal family of serine protease inhibitors. SPARC, however, does not appear to have intrinsic protease inhibitor activity. The C-terminal domain of SPARC contains an extracellular EF-hand structure that confers collagen binding via the helix aA at residues 149–156 (Hohenester et al., 1996; Hohenester, Maurer, & Timpl, 1997). The collagen-binding pocket formed by SPARC closely resembles that of the collagen-binding discoidin domain receptors (DDR1 and DDR2), although the primary amino acid structure of these distinct collagen-binding proteins differs, an example of convergent evolution of collagen-binding domains (Hohenester, Sasaki, Giudici, Farndale, & Bachinger, 2008). Hence, the binding site on fibrillar collagen is predicted to be the same sequence for SPARC and for the collagen receptors DDR1 and 2. SPARC and DDR receptors are expected to compete with one another for collagen binding. Sasaki et al. demonstrated that recombinant SPARC produced with a deletion of amino acids 196–203, representing the helix aC in the EF hand, resulted in a form of SPARC protein that bound fibrillar collagens with a 10-fold increase in affinity in comparison to full-length SPARC (Sasaki et al., 1997; Sasaki, Hohenester, Gohring, & Timpl, 1998). The helix aC is predicted to partially interfere with binding of collagen via helix aA. Thus, removing helix aC, either by recombinant expression or perhaps by protease digestion of native SPARC in the extracellular space, results in a form of SPARC with higher binding affinity for collagens. SPARC has 14 cysteine residues that are predicted to form 7 intramolecular disulfide bonds to generate the distinct tertiary structure of this matricellular protein. Hence, when proteins are separated by SDS-PAGE analysis, the addition of reducing agents will cause a shift in the MW of SPARC due to the release of the disulfide intramolecular bonds (Mason, Taylor, Williams, Sage, & Hogan, 1986). SPARC protein separated under nonreducing conditions will migrate more quickly than SPARC protein separated under reducing conditions due to the fact that unfolded SPARC with disulfide bonds released will migrate more slowly. SPARC undergoes N-glycosylation on asparagine (aa 99) during posttranslation modification
2 Method
(Hughes, Taylor, Sage, & Hogan, 1987). Differential glycosylation of SPARC has been implicated in collagen-binding properties of SPARC. For example, SPARC derived from bone, that has high mannose structures and biantennary structures, binds collagen with higher affinity than SPARC purified from platelets that has biantennary and triantennary complex structures (Kelm & Mann, 1991). Thus, cell-type-specific production of SPARC can give rise to different forms of glycosylated SPARC with functional differences.
2 METHOD 2.1 BACKGROUND Production of recombinant SPARC has been reported in E. coli, yeast, insect cells, and mammalian cells (Bassuk, Baneyx, Vernon, Funk, & Sage, 1996; Bradshaw, Bassuk, Francki, & Sage, 2000; Hohenadl et al., 1995; Nischt et al., 1991; Yost, Bell, Seale, & Sage, 1994). Given that differential glycosylation of SPARC can influence collagen-binding properties, expression systems such as bacteria and yeast might not be ideal for production of SPARC for use in assays of extracellular matrix assembly. In addition, SPARC produced in E. coli tends to form insoluble aggregates (Schneider, Thomas, Bassuk, Sage, & Baneyx, 1997). Notably, recombinant SPARC produced in bacteria and in yeast was, however, shown to elicit changes in cell proliferation and cell attachment, suggesting that glycosylation by mammalian enzymes is not required for these functional properties of SPARC (Bassuk et al., 1996; Yost et al., 1994). Production of SPARC in mammalian cells yields glycosylated SPARC; however, attention to differential modification of SPARC from distinct cell types should be taken into account. SPARC produced by the tumor-derived Engelbreth-HolmSwarm (EHS) cells, for example, is likely to have different glycosylation than that produced by cells of osteoblastic origin and consequently might bind differently to collagens. In addition, SPARC produced in mammalian systems has the disadvantage that proteins that copurify with SPARC are predicted to have the potential to elicit confounding effects on mammalian cells as opposed to bacterial SPARC or SPARC produced by insect cells. Hence, recombinant SPARC expression driven by baculoviral expression in insect cells was adopted as a method of choice for producing large amounts of biologically active SPARC for assays involving mammalian cells and systems.
2.2 INSECT CELL CULTURE Insect cells: Spodoptera frugiperda IPLB-SF21-AE (Sf9 cells in SF-900™ III SFM, Thermo Fisher) SF-900™ III serum-free media (Thermo Fisher) 200 mM L-Glutamine
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Frozen insect cells are initiated in either static or shaker culture. One vial of frozen cells is resuspended in 27 mL of media, placed in a 75 cm2 flask (for stationary cultures), and placed in an incubator at 27°C; or placed in a 125 mL spinner flask in a shaking water bath at 27°C. When initiating insect cell cultures from frozen stocks, it is not necessary, and can be detrimental, to spin the cells to remove the freezing media. For spinner cultures, allow the cell density to reach 2 million cells per mL of media, then add an additional 25 mL of media for a total of 50 mL. After the cells double, move them to a 250 mL spinner (100 mL total cell suspension), still in the shaking water bath. After the cells double again, maintain them as a spinner culture, expanding them to 200 mL in a 500 mL spinner flask. Cryopreservation of low-passage cells at 10 million cells per mL can be performed for later use. Penicillin–streptomycin and fungizone are routinely added to the media during the initiation of insect cell cultures. Once cells are established and cryopreserved, the antibiotic and antimycotic solutions can be eliminated. Contamination of cultures generally signals poor conditions in the shaking water bath or incubator where cells are cultured. Cleaning water baths and incubators once every 3 months is recommended and should be sufficient to prevent contamination. Insect cells can be routinely subcultured to as low as 0.5 million cells per mL. They should be passaged before they exceed 4 million cells per mL. If cell density exceeds 4 million per mL, then split the cells to 1.5 million per mL and subculture as usual the following day. For stationary cultures, cells are routinely monitored for degree of confluence. Cells should be passaged when cultures approach 80%–90% confluency. It is recommended to subculture cells in a 1:2 or 1:3 ratio; higher dilution compromise cell viability. For large-scale production, insect cells can be grown in large flasks such as T175s and T225s (Thermo Fisher).
2.3 BACULOVIRAL EXPRESSION OF HUMAN SPARC A baculovirus infector encoding SPARC cDNA is generated using an appropriate baculoviral kit, e.g., the Bac-to-Bac® Baculovirus Expression System (Thermo Fisher). Follow kit instructions to generate the baculovirus infector to drive recombinant SPARC expression. It is important to empirically determine optimal amounts of infector for each newly generated preparation of baculovirus. To this end, plate insect cells in a 12-well plate at 3.3 105/well in 1 mL of media (no FBS). Add the new infector starting at 1 mL/well and titrate down 1:2. After 5 days, collect media from each of the wells, spin, and determine levels of SPARC by ELISA or by western blot. To be within range of an ELISA, the conditioned media will need to be titrated at 1:100, 1:1000, and 1:10,000. Transduce insect cells with baculovirus expressing SPARC using the empirically derived amount. The smallest volume that efficiently infects the insect cells should be used. Infection should be initiated when the cells are in a doubling phase and have reached 2 million cells per mL (in spinner culture) or 50%–60% confluence for stationary cultures. Cell viability must be greater than 90%. Infect 400 mL of cell
3 Purification of recombinant SPARC produced by insect cells
suspension per 2 L Erlenmeyer flask in a shaking water bath at 27°C set to 150 rpm. Spinner flasks on spin plates may also be used, though the productivity may be lower. Production of SPARC in large tissue culture flasks (T175 or T225) under static conditions is a viable option in the event that a shaking water bath or spinner flask apparatus is not available. Flasks should be incubated at 27°C for efficient recombinant protein production. When cell viability declines to 75% or less (usually day 4 postinfection), spin the cell solution at low speed until the supernatant is clarified. Infected cells generally exhibit large vacuoles indicative of recombinant protein production. Collect the supernatant and add 0.1 M PMSF in 95% ethanol at 1:500 dropwise while stirring (to inhibit proteases). Filter the supernatant with a 0.22 mm bottle top filter (1 filter per 400 mL). The option to add 0.02% sodium azide to protect the conditioned media from contamination during SPARC purification can be implemented.
2.4 MAINTENANCE OF VIRAL VECTOR Viral infector remains potent for about 6 months. When production of SPARC begins to diminish, it is recommended to generate new viral infector. Plate insect cells at 5 106 cells per 10 cm dish in 15 mL Sf9 III media with 10% FBS. It is useful to prepare at least six dishes. Titrate down from the volume of infector previously used for optimal production of recombinant SPARC. Established infector concentrations routinely range from 1 to 5 mL per 5 106 cells. Titrate the infector in the tissue culture dishes as follows: 0.5x, 0.1x, 0.05x, 0.01x, and no infector (negative control), where x is the optimal infector concentration previously used for production of recombinant SPARC. The objective is to find the lowest concentration of virus that delivers maximal infection rates. Incubate the 10 cm dishes in a loosely covered container at 27°C with hydration. Check cells 4 days postaddition of infector for signs of infection—enlarged cells with vacuoles and floaters. Select the dish with the lowest level of virus that clearly resulted in infection. Spin the contents of the dish and retain the viral supernatant as new infector stock. Do not filter the infector stock. Store the infector at 4°C. The greater the number of infector generations, the more likely mutations will accumulate. Monitor the quality of recombinant SPARC produced, and when it degrades, initiate the generation of new baculovirus encoding SPARC DNA as before.
3 PURIFICATION OF RECOMBINANT SPARC PRODUCED BY INSECT CELLS 3.1 BACKGROUND Recombinant production of SPARC in insect cells was originally described to involve a two-stage purification scheme: purification by size-exclusion chromatography followed by ion-exchange chromatography (Bradshaw et al., 2000). Since this
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report, we have optimized production and purification using a single ion-exchange chromatography system performed on a larger scale to yield increased amounts of recombinant protein. The single step significantly reduces time and expense associated with recombinant SPARC production. In addition, the intrinsic labile nature of SPARC protein was not compatible with a multistep process. Frequently, preparations requiring multiple steps resulted in recombinant protein with reduced biological activity. Thus, whereas the purity of recombinant SPARC might be slightly compromised by the one-step purification, the quality of SPARC protein in terms of biological activity is significantly improved.
3.2 EXPERIMENTAL METHOD Chromatography buffers: Starting buffer (20 mM MOPS, 200 mM LiCl, pH 6.5) 200 mM MOPS 5 M LiCl dd H2O Adjust pH to 6.5 Q.S. to 2 L
200 mL 80 mL 1700 mL
Elution buffer (20 mM MOPS, 400 mM LiCl, pH 6.5) 200 mM MOPS 5 M LiCl dd H2O Adjust pH to 6.5 Q.S. to 500 mL
50 mL 40 mL 400 mL
Wash buffer (20 mM MOPS, 2 M LiCl) 200 mM MOPS 5 M LiCl dd H2O Q.S. to 500 mL
50 mL 200 mL
Chromatography equipment can be used either at room temperature or at 4°C if available. If chromatography is performed at room temperature, keep starting buffer, elution buffer, and conditioned media containing recombinant SPARC chilled.
3 Purification of recombinant SPARC produced by insect cells
Preparing the column: Add water to a clean column (e.g., Amersham Biosciences XK50) with the outflow in the closed position to a height of 2–3 cm. Pour 50 mL of resuspended Q Sepharose Fast Flow (GE Healthcare Life Sciences) into the column. Allow gel to settle before completing the column assembly. Connect column to, for € example, an AKTAPrime plus automated liquid chromatography system (GE Healthcare Life Sciences) or similar automated column assembly, preferably one with a spectrophotometer unit (A280) to monitor protein flow through the column. Pack column with 3 column bed volumes of water at 15 mL/min. Equilibrate column at 10 mL/min with 2 bed volumes of starting buffer. Check to ensure that pH of outflow is 6.5. If necessary, continue equilibrating the column until the outflow reaches a pH of 6.5. Purifying SPARC from conditioned media: Combine conditioned media containing SPARC with 1/10 volume of 200 mM MOPS pH 6.5. Adjust pH of mixture to 6.5. Typically, 400–500 mL of conditioned media are used for one preparation. Load the buffered conditioned media onto the column at 10 mL/min. Once the conditioned media are loaded onto the column, wash the column at 10 mL/min with starting buffer until minimal protein is detected in the outflow either using the A280 readout from the chromatography system or by monitoring individual fractions on a spectrophotometer. SPARC is eluted on a continuous salt gradient created with 150 mL of starting buffer and 150 mL of elution buffer. The gradient can be established using a gradient maker or using an automated system. The gradient should run from 0% to 100% elution buffer at 5 mL/min. It is recommended to collect 3.5 mL fractions. SPARC protein will elute during the gradient and is detected by a spike in the A280 readout. Once the gradient has completed and run through the column, the column can be stripped of all remaining proteins with wash buffer at 5 mL/min. Store column matrix in running buffer containing 0.02% sodium azide. Typically, matrices in columns are used for 4–5 runs and then replaced with fresh Q-Sepharose.
3.3 ANALYSIS AND STORAGE OF RECOMBINANT SPARC Fractions eluted during the salt gradient with peak A280 values are analyzed by SDSPAGE on 10% gels (Fig. 1). Fractions containing SPARC are combined and concentrated to 1–5 mg/mL in a Centricon Plus-70 (30,000 MWCO, EMD Millipore). Recombinant SPARC can then be dialyzed against a suitable buffer of choice such as PBS or Hank’s balanced salt solution amenable for downstream applications (the addition of CaCl2 to 2 mM is recommended to retain SPARC structural integrity). It is recommended to divide purified SPARC into appropriate aliquots and snap freeze in liquid nitrogen for storage at 80°C. Purified SPARC rapidly loses biological activity when stored at 4°C or when subjected to freeze–thaw cycles. Several small aliquots of recombinant protein may be set aside for initial testing of purification and activity.
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FIG. 1 Purification of recombinant human SPARC from baculovirus-infected insect cells. (A) Coomassie of fractions 46–53 eluted from a Q-Sepharose ion-exchange column using a salt gradient and separated by SDS-PAGE under nonreducing conditions. (B) Fractions 46–53 separated by SDS-PAGE and blotted with antibodies raised against human SPARC. Migration of molecular weight markers is indicated on the left.
Routinely, the purity of recombinant SPARC preparations is estimated by separation using SDS-PAGE. For example, 5 mg of purified protein is separated on a 10% gel and stained with Coomassie blue (Fig. 2). Generally, SPARC will be about 80%–90% pure; therefore, purity would equal 0.8–0.9. The final concentration of SPARC can then be determined by the following calculation: ðA280 purityÞ=extinction coefficient 0:915 ¼ mg SPARC=mL Yield should be 4–7 mg SPARC per 400 mL of Sf9 cell conditioned media. Testing for SPARC functional activity: Purified recombinant SPARC activity is routinely assessed in assays of cell proliferation as SPARC has been shown to inhibit cell proliferation in a number of different cell types. For example, bovine aortic endothelial (BAE) cells are treated with 5, 10, and 20 mg SPARC/mL; cell proliferation is monitored using tritiated thymidine incorporation or a similar assay of proliferation. Any given preparation of recombinant SPARC will possibly contain some endotoxin activity originating from the Sf9 media. Endotoxin is particularly
References
FIG. 2 Purified recombinant SPARC protein dialyzed and concentrated for functional analyses separated by SDS-PAGE and stained with Coomassie blue. Migration of molecular weight markers are shown on the left. Recombinant SPARC protein purified from conditioned insect cell media eluted from an ion-exchange column using a salt gradient yields SPARC protein that is 90% pure.
problematic when SPARC activity on endothelial cell function is being assessed. Use of a kit for detection of endotoxin (e.g., Lonza or Thermo Fisher) is recommended to determine whether the endotoxin content of the SPARC preparation exceeds the expected concentration. Less than 10 ng endotoxin per mg SPARC is optimal. For example, in an assay of cell proliferation using BAE cells treated with 60 mg/mL recombinant SPARC from a preparation containing 10 ng of endotoxin per mg of SPARC, typically less than 10% of the inhibition of proliferation is attributable to endotoxin activity. To determine the effect of endotoxin on other types of cells, treat the designated cells with a titration of control standard endotoxin (a component of the endotoxin kit) and monitor cell proliferation. Additional tests of functional activity for recombinant SPARC include cell-rounding assays and collagen-binding analysis (Sage, Vernon, Funk, Everitt, & Angello, 1989; Sasaki et al., 1997).
REFERENCES Bassuk, J. A., Baneyx, F., Vernon, R. B., Funk, S. E., & Sage, E. H. (1996). Expression of biologically active human SPARC in Escherichia coli. Archives of Biochemistry and Biophysics, 325(1), 8–19. Bradshaw, A. D. (2012). Diverse biological functions of the SPARC family of proteins. The International Journal of Biochemistry & Cell Biology, 44(3), 480–488. S1357-2725(12) 00004-0 [pii]. https://doi.org/10.1016/j.biocel.2011.12.021. Bradshaw, A. D., Bassuk, J. A., Francki, A., & Sage, E. H. (2000). Expression and purification of recombinant human SPARC produced by baculovirus. Molecular Cell Biology Research Communications, 3(6), 345–351. Brekken, R. A., & Sage, E. H. (2001). SPARC, a matricellular protein: At the crossroads of cell-matrix communication. Matrix Biology, 19(8), 816–827.
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Hohenadl, C., Mann, K., Mayer, U., Timpl, R., Paulsson, M., & Aeschlimann, D. (1995). Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification. The Journal of Biological Chemistry, 270(40), 23415–23420. Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., & Engel, J. (1996). Structure of a novel extracellular Ca(2 +)-binding module in BM-40. Nature Structural Biology, 3(1), 67–73. Hohenester, E., Maurer, P., & Timpl, R. (1997). Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40. The EMBO Journal, 16(13), 3778–3786. Hohenester, E., Sasaki, T., Giudici, C., Farndale, R. W., & Bachinger, H. P. (2008). Structural basis of sequence-specific collagen recognition by SPARC. Proceedings of the National Academy of Sciences of the United States of America, 105(47), 18273–18277. Hughes, R. C., Taylor, A., Sage, H., & Hogan, B. L. (1987). Distinct patterns of glycosylation of colligin, a collagen-binding glycoprotein, and SPARC (osteonectin), a secreted Ca2 +-binding glycoprotein. Evidence for the localisation of colligin in the endoplasmic reticulum. European Journal of Biochemistry, 163(1), 57–65. Kelm, R. J., Jr., & Mann, K. G. (1991). The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation. The Journal of Biological Chemistry, 266(15), 9632–9639. Mason, I. J., Taylor, A., Williams, J. G., Sage, H., & Hogan, B. L. (1986). Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell ‘culture shock’ glycoprotein of Mr 43,000. The EMBO Journal, 5(7), 1465–1472. Mendoza-Londono, R., Fahiminiya, S., Majewski, J., Tetreault, M., Nadaf, J., Kannu, P., et al. (2015). Recessive osteogenesis imperfecta caused by missense mutations in SPARC. American Journal of Human Genetics, 96(6), 979–985. https://doi.org/10.1016/j. ajhg.2015.04.021. Nischt, R., Pottgiesser, J., Krieg, T., Mayer, U., Aumailley, M., & Timpl, R. (1991). Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40. European Journal of Biochemistry, 200(2), 529–536. Rosset, E. M., & Bradshaw, A. D. (2016). SPARC/osteonectin in mineralized tissue. Matrix Biology, 52–54, 78–87. https://doi.org/10.1016/j.matbio.2016.02.001. Sage, H., Vernon, R. B., Funk, S. E., Everitt, E. A., & Angello, J. (1989). SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca +2-dependent binding to the extracellular matrix. The Journal of Cell Biology, 109(1), 341–356. Sasaki, T., Gohring, W., Mann, K., Maurer, P., Hohenester, E., Knauper, V., et al. (1997). Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens. The Journal of Biological Chemistry, 272(14), 9237–9243. Sasaki, T., Hohenester, E., Gohring, W., & Timpl, R. (1998). Crystal structure and mapping by site-directed mutagenesis of the collagen-binding epitope of an activated form of BM-40/ SPARC/osteonectin. The EMBO Journal, 17(6), 1625–1634. Schneider, E. L., Thomas, J. G., Bassuk, J. A., Sage, E. H., & Baneyx, F. (1997). Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli. Nature Biotechnology, 15(6), 581–585. https://doi.org/10.1038/nbt0697-581.
References
Trombetta-Esilva, J., & Bradshaw, A. D. (2012). The function of SPARC as a mediator of fibrosis. The Open Rheumatology Journal, 6, 146–155. https://doi.org/10.2174/ 1874312901206010146. Yost, J. C., Bell, A., Seale, R., & Sage, E. H. (1994). Purification of biologically active SPARC expressed in Saccharomyces cerevisiae. Archives of Biochemistry and Biophysics, 314(1), 50–63.
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Production and purification of recombinant human SPARC
Gail Workman*, Amy D. Bradshaw†,‡,1 *Matrix Biology Program, Benaroya Research Institute, Seattle, WA, United States Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, SC, United States ‡ Ralph H. Johnson Department of Veteran’s Affairs Medical Center, Charleston, SC, United States 1 Corresponding author: e-mail address: [email protected] †
CHAPTER OUTLINE 1 Introduction ........................................................................................................336 2 Method...............................................................................................................337 2.1 Background ........................................................................................337 2.2 Insect Cell Culture...............................................................................337 2.3 Baculoviral Expression of Human SPARC ..............................................338 2.4 Maintenance of Viral Vector..................................................................339 3 Purification of Recombinant SPARC Produced by Insect Cells ................................339 3.1 Background ........................................................................................339 3.2 Experimental Method...........................................................................340 3.3 Analysis and Storage of Recombinant SPARC ........................................341 References ............................................................................................................. 343
Abstract The matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or as BM-40) is a collagen-binding protein with a capacity to induce cell rounding and influence proliferation in cultured cells. In mice that do not express SPARC, fibrillar collagen is reduced in some adult tissues; notably, a reduction in fibrosis is reported in response to fibrotic stimuli in lungs, heart, skin, liver, and in the eye. Recently, mutations in the gene encoding SPARC were found in patients afflicted with osteogenesis imperfecta. Thus, SPARC appears to be a critical mediator of collagen deposition and assembly in tissues. A useful tool for assessing the function of SPARC in ECM assembly is a source of purified recombinant SPARC. Outlined in this chapter is a brief discussion of different strategies for generating recombinant SPARC and an experimental strategy for producing and purifying human recombinant SPARC driven by baculoviral expression in insect cells. Methods in Cell Biology, Volume 143, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2017.08.020 © 2018 Elsevier Inc. All rights reserved.
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1 INTRODUCTION SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein involved in tissue remodeling and fibrosis (Bradshaw, 2012; Trombetta-Esilva & Bradshaw, 2012). Mutations in the gene encoding SPARC were recently associated with a type of osteogenesis imperfecta, a finding that reinforces the hypothesis that SPARC is critical for collagen deposition in mineralized tissues as well as in nonmineralized tissues (Mendoza-Londono et al., 2015; Rosset & Bradshaw, 2016). Typical of most matricellular proteins, SPARC has a modular structure. SPARC has three domains (Brekken & Sage, 2001). The N-terminal domain contains a high-capacity, low-affinity Ca2+-binding site, with a sequence located in the first 10 amino acids that serves as a transglutaminase recognition site. The second domain is characterized by a follistatin-like domain with similarities to the Kazal family of serine protease inhibitors. SPARC, however, does not appear to have intrinsic protease inhibitor activity. The C-terminal domain of SPARC contains an extracellular EF-hand structure that confers collagen binding via the helix aA at residues 149–156 (Hohenester et al., 1996; Hohenester, Maurer, & Timpl, 1997). The collagen-binding pocket formed by SPARC closely resembles that of the collagen-binding discoidin domain receptors (DDR1 and DDR2), although the primary amino acid structure of these distinct collagen-binding proteins differs, an example of convergent evolution of collagen-binding domains (Hohenester, Sasaki, Giudici, Farndale, & Bachinger, 2008). Hence, the binding site on fibrillar collagen is predicted to be the same sequence for SPARC and for the collagen receptors DDR1 and 2. SPARC and DDR receptors are expected to compete with one another for collagen binding. Sasaki et al. demonstrated that recombinant SPARC produced with a deletion of amino acids 196–203, representing the helix aC in the EF hand, resulted in a form of SPARC protein that bound fibrillar collagens with a 10-fold increase in affinity in comparison to full-length SPARC (Sasaki et al., 1997; Sasaki, Hohenester, Gohring, & Timpl, 1998). The helix aC is predicted to partially interfere with binding of collagen via helix aA. Thus, removing helix aC, either by recombinant expression or perhaps by protease digestion of native SPARC in the extracellular space, results in a form of SPARC with higher binding affinity for collagens. SPARC has 14 cysteine residues that are predicted to form 7 intramolecular disulfide bonds to generate the distinct tertiary structure of this matricellular protein. Hence, when proteins are separated by SDS-PAGE analysis, the addition of reducing agents will cause a shift in the MW of SPARC due to the release of the disulfide intramolecular bonds (Mason, Taylor, Williams, Sage, & Hogan, 1986). SPARC protein separated under nonreducing conditions will migrate more quickly than SPARC protein separated under reducing conditions due to the fact that unfolded SPARC with disulfide bonds released will migrate more slowly. SPARC undergoes N-glycosylation on asparagine (aa 99) during posttranslation modification
2 Method
(Hughes, Taylor, Sage, & Hogan, 1987). Differential glycosylation of SPARC has been implicated in collagen-binding properties of SPARC. For example, SPARC derived from bone, that has high mannose structures and biantennary structures, binds collagen with higher affinity than SPARC purified from platelets that has biantennary and triantennary complex structures (Kelm & Mann, 1991). Thus, cell-type-specific production of SPARC can give rise to different forms of glycosylated SPARC with functional differences.
2 METHOD 2.1 BACKGROUND Production of recombinant SPARC has been reported in E. coli, yeast, insect cells, and mammalian cells (Bassuk, Baneyx, Vernon, Funk, & Sage, 1996; Bradshaw, Bassuk, Francki, & Sage, 2000; Hohenadl et al., 1995; Nischt et al., 1991; Yost, Bell, Seale, & Sage, 1994). Given that differential glycosylation of SPARC can influence collagen-binding properties, expression systems such as bacteria and yeast might not be ideal for production of SPARC for use in assays of extracellular matrix assembly. In addition, SPARC produced in E. coli tends to form insoluble aggregates (Schneider, Thomas, Bassuk, Sage, & Baneyx, 1997). Notably, recombinant SPARC produced in bacteria and in yeast was, however, shown to elicit changes in cell proliferation and cell attachment, suggesting that glycosylation by mammalian enzymes is not required for these functional properties of SPARC (Bassuk et al., 1996; Yost et al., 1994). Production of SPARC in mammalian cells yields glycosylated SPARC; however, attention to differential modification of SPARC from distinct cell types should be taken into account. SPARC produced by the tumor-derived Engelbreth-HolmSwarm (EHS) cells, for example, is likely to have different glycosylation than that produced by cells of osteoblastic origin and consequently might bind differently to collagens. In addition, SPARC produced in mammalian systems has the disadvantage that proteins that copurify with SPARC are predicted to have the potential to elicit confounding effects on mammalian cells as opposed to bacterial SPARC or SPARC produced by insect cells. Hence, recombinant SPARC expression driven by baculoviral expression in insect cells was adopted as a method of choice for producing large amounts of biologically active SPARC for assays involving mammalian cells and systems.
2.2 INSECT CELL CULTURE Insect cells: Spodoptera frugiperda IPLB-SF21-AE (Sf9 cells in SF-900™ III SFM, Thermo Fisher) SF-900™ III serum-free media (Thermo Fisher) 200 mM L-Glutamine
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Frozen insect cells are initiated in either static or shaker culture. One vial of frozen cells is resuspended in 27 mL of media, placed in a 75 cm2 flask (for stationary cultures), and placed in an incubator at 27°C; or placed in a 125 mL spinner flask in a shaking water bath at 27°C. When initiating insect cell cultures from frozen stocks, it is not necessary, and can be detrimental, to spin the cells to remove the freezing media. For spinner cultures, allow the cell density to reach 2 million cells per mL of media, then add an additional 25 mL of media for a total of 50 mL. After the cells double, move them to a 250 mL spinner (100 mL total cell suspension), still in the shaking water bath. After the cells double again, maintain them as a spinner culture, expanding them to 200 mL in a 500 mL spinner flask. Cryopreservation of low-passage cells at 10 million cells per mL can be performed for later use. Penicillin–streptomycin and fungizone are routinely added to the media during the initiation of insect cell cultures. Once cells are established and cryopreserved, the antibiotic and antimycotic solutions can be eliminated. Contamination of cultures generally signals poor conditions in the shaking water bath or incubator where cells are cultured. Cleaning water baths and incubators once every 3 months is recommended and should be sufficient to prevent contamination. Insect cells can be routinely subcultured to as low as 0.5 million cells per mL. They should be passaged before they exceed 4 million cells per mL. If cell density exceeds 4 million per mL, then split the cells to 1.5 million per mL and subculture as usual the following day. For stationary cultures, cells are routinely monitored for degree of confluence. Cells should be passaged when cultures approach 80%–90% confluency. It is recommended to subculture cells in a 1:2 or 1:3 ratio; higher dilution compromise cell viability. For large-scale production, insect cells can be grown in large flasks such as T175s and T225s (Thermo Fisher).
2.3 BACULOVIRAL EXPRESSION OF HUMAN SPARC A baculovirus infector encoding SPARC cDNA is generated using an appropriate baculoviral kit, e.g., the Bac-to-Bac® Baculovirus Expression System (Thermo Fisher). Follow kit instructions to generate the baculovirus infector to drive recombinant SPARC expression. It is important to empirically determine optimal amounts of infector for each newly generated preparation of baculovirus. To this end, plate insect cells in a 12-well plate at 3.3 105/well in 1 mL of media (no FBS). Add the new infector starting at 1 mL/well and titrate down 1:2. After 5 days, collect media from each of the wells, spin, and determine levels of SPARC by ELISA or by western blot. To be within range of an ELISA, the conditioned media will need to be titrated at 1:100, 1:1000, and 1:10,000. Transduce insect cells with baculovirus expressing SPARC using the empirically derived amount. The smallest volume that efficiently infects the insect cells should be used. Infection should be initiated when the cells are in a doubling phase and have reached 2 million cells per mL (in spinner culture) or 50%–60% confluence for stationary cultures. Cell viability must be greater than 90%. Infect 400 mL of cell
3 Purification of recombinant SPARC produced by insect cells
suspension per 2 L Erlenmeyer flask in a shaking water bath at 27°C set to 150 rpm. Spinner flasks on spin plates may also be used, though the productivity may be lower. Production of SPARC in large tissue culture flasks (T175 or T225) under static conditions is a viable option in the event that a shaking water bath or spinner flask apparatus is not available. Flasks should be incubated at 27°C for efficient recombinant protein production. When cell viability declines to 75% or less (usually day 4 postinfection), spin the cell solution at low speed until the supernatant is clarified. Infected cells generally exhibit large vacuoles indicative of recombinant protein production. Collect the supernatant and add 0.1 M PMSF in 95% ethanol at 1:500 dropwise while stirring (to inhibit proteases). Filter the supernatant with a 0.22 mm bottle top filter (1 filter per 400 mL). The option to add 0.02% sodium azide to protect the conditioned media from contamination during SPARC purification can be implemented.
2.4 MAINTENANCE OF VIRAL VECTOR Viral infector remains potent for about 6 months. When production of SPARC begins to diminish, it is recommended to generate new viral infector. Plate insect cells at 5 106 cells per 10 cm dish in 15 mL Sf9 III media with 10% FBS. It is useful to prepare at least six dishes. Titrate down from the volume of infector previously used for optimal production of recombinant SPARC. Established infector concentrations routinely range from 1 to 5 mL per 5 106 cells. Titrate the infector in the tissue culture dishes as follows: 0.5x, 0.1x, 0.05x, 0.01x, and no infector (negative control), where x is the optimal infector concentration previously used for production of recombinant SPARC. The objective is to find the lowest concentration of virus that delivers maximal infection rates. Incubate the 10 cm dishes in a loosely covered container at 27°C with hydration. Check cells 4 days postaddition of infector for signs of infection—enlarged cells with vacuoles and floaters. Select the dish with the lowest level of virus that clearly resulted in infection. Spin the contents of the dish and retain the viral supernatant as new infector stock. Do not filter the infector stock. Store the infector at 4°C. The greater the number of infector generations, the more likely mutations will accumulate. Monitor the quality of recombinant SPARC produced, and when it degrades, initiate the generation of new baculovirus encoding SPARC DNA as before.
3 PURIFICATION OF RECOMBINANT SPARC PRODUCED BY INSECT CELLS 3.1 BACKGROUND Recombinant production of SPARC in insect cells was originally described to involve a two-stage purification scheme: purification by size-exclusion chromatography followed by ion-exchange chromatography (Bradshaw et al., 2000). Since this
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report, we have optimized production and purification using a single ion-exchange chromatography system performed on a larger scale to yield increased amounts of recombinant protein. The single step significantly reduces time and expense associated with recombinant SPARC production. In addition, the intrinsic labile nature of SPARC protein was not compatible with a multistep process. Frequently, preparations requiring multiple steps resulted in recombinant protein with reduced biological activity. Thus, whereas the purity of recombinant SPARC might be slightly compromised by the one-step purification, the quality of SPARC protein in terms of biological activity is significantly improved.
3.2 EXPERIMENTAL METHOD Chromatography buffers: Starting buffer (20 mM MOPS, 200 mM LiCl, pH 6.5) 200 mM MOPS 5 M LiCl dd H2O Adjust pH to 6.5 Q.S. to 2 L
200 mL 80 mL 1700 mL
Elution buffer (20 mM MOPS, 400 mM LiCl, pH 6.5) 200 mM MOPS 5 M LiCl dd H2O Adjust pH to 6.5 Q.S. to 500 mL
50 mL 40 mL 400 mL
Wash buffer (20 mM MOPS, 2 M LiCl) 200 mM MOPS 5 M LiCl dd H2O Q.S. to 500 mL
50 mL 200 mL
Chromatography equipment can be used either at room temperature or at 4°C if available. If chromatography is performed at room temperature, keep starting buffer, elution buffer, and conditioned media containing recombinant SPARC chilled.
3 Purification of recombinant SPARC produced by insect cells
Preparing the column: Add water to a clean column (e.g., Amersham Biosciences XK50) with the outflow in the closed position to a height of 2–3 cm. Pour 50 mL of resuspended Q Sepharose Fast Flow (GE Healthcare Life Sciences) into the column. Allow gel to settle before completing the column assembly. Connect column to, for € example, an AKTAPrime plus automated liquid chromatography system (GE Healthcare Life Sciences) or similar automated column assembly, preferably one with a spectrophotometer unit (A280) to monitor protein flow through the column. Pack column with 3 column bed volumes of water at 15 mL/min. Equilibrate column at 10 mL/min with 2 bed volumes of starting buffer. Check to ensure that pH of outflow is 6.5. If necessary, continue equilibrating the column until the outflow reaches a pH of 6.5. Purifying SPARC from conditioned media: Combine conditioned media containing SPARC with 1/10 volume of 200 mM MOPS pH 6.5. Adjust pH of mixture to 6.5. Typically, 400–500 mL of conditioned media are used for one preparation. Load the buffered conditioned media onto the column at 10 mL/min. Once the conditioned media are loaded onto the column, wash the column at 10 mL/min with starting buffer until minimal protein is detected in the outflow either using the A280 readout from the chromatography system or by monitoring individual fractions on a spectrophotometer. SPARC is eluted on a continuous salt gradient created with 150 mL of starting buffer and 150 mL of elution buffer. The gradient can be established using a gradient maker or using an automated system. The gradient should run from 0% to 100% elution buffer at 5 mL/min. It is recommended to collect 3.5 mL fractions. SPARC protein will elute during the gradient and is detected by a spike in the A280 readout. Once the gradient has completed and run through the column, the column can be stripped of all remaining proteins with wash buffer at 5 mL/min. Store column matrix in running buffer containing 0.02% sodium azide. Typically, matrices in columns are used for 4–5 runs and then replaced with fresh Q-Sepharose.
3.3 ANALYSIS AND STORAGE OF RECOMBINANT SPARC Fractions eluted during the salt gradient with peak A280 values are analyzed by SDSPAGE on 10% gels (Fig. 1). Fractions containing SPARC are combined and concentrated to 1–5 mg/mL in a Centricon Plus-70 (30,000 MWCO, EMD Millipore). Recombinant SPARC can then be dialyzed against a suitable buffer of choice such as PBS or Hank’s balanced salt solution amenable for downstream applications (the addition of CaCl2 to 2 mM is recommended to retain SPARC structural integrity). It is recommended to divide purified SPARC into appropriate aliquots and snap freeze in liquid nitrogen for storage at 80°C. Purified SPARC rapidly loses biological activity when stored at 4°C or when subjected to freeze–thaw cycles. Several small aliquots of recombinant protein may be set aside for initial testing of purification and activity.
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FIG. 1 Purification of recombinant human SPARC from baculovirus-infected insect cells. (A) Coomassie of fractions 46–53 eluted from a Q-Sepharose ion-exchange column using a salt gradient and separated by SDS-PAGE under nonreducing conditions. (B) Fractions 46–53 separated by SDS-PAGE and blotted with antibodies raised against human SPARC. Migration of molecular weight markers is indicated on the left.
Routinely, the purity of recombinant SPARC preparations is estimated by separation using SDS-PAGE. For example, 5 mg of purified protein is separated on a 10% gel and stained with Coomassie blue (Fig. 2). Generally, SPARC will be about 80%–90% pure; therefore, purity would equal 0.8–0.9. The final concentration of SPARC can then be determined by the following calculation: ðA280 purityÞ=extinction coefficient 0:915 ¼ mg SPARC=mL Yield should be 4–7 mg SPARC per 400 mL of Sf9 cell conditioned media. Testing for SPARC functional activity: Purified recombinant SPARC activity is routinely assessed in assays of cell proliferation as SPARC has been shown to inhibit cell proliferation in a number of different cell types. For example, bovine aortic endothelial (BAE) cells are treated with 5, 10, and 20 mg SPARC/mL; cell proliferation is monitored using tritiated thymidine incorporation or a similar assay of proliferation. Any given preparation of recombinant SPARC will possibly contain some endotoxin activity originating from the Sf9 media. Endotoxin is particularly
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FIG. 2 Purified recombinant SPARC protein dialyzed and concentrated for functional analyses separated by SDS-PAGE and stained with Coomassie blue. Migration of molecular weight markers are shown on the left. Recombinant SPARC protein purified from conditioned insect cell media eluted from an ion-exchange column using a salt gradient yields SPARC protein that is 90% pure.
problematic when SPARC activity on endothelial cell function is being assessed. Use of a kit for detection of endotoxin (e.g., Lonza or Thermo Fisher) is recommended to determine whether the endotoxin content of the SPARC preparation exceeds the expected concentration. Less than 10 ng endotoxin per mg SPARC is optimal. For example, in an assay of cell proliferation using BAE cells treated with 60 mg/mL recombinant SPARC from a preparation containing 10 ng of endotoxin per mg of SPARC, typically less than 10% of the inhibition of proliferation is attributable to endotoxin activity. To determine the effect of endotoxin on other types of cells, treat the designated cells with a titration of control standard endotoxin (a component of the endotoxin kit) and monitor cell proliferation. Additional tests of functional activity for recombinant SPARC include cell-rounding assays and collagen-binding analysis (Sage, Vernon, Funk, Everitt, & Angello, 1989; Sasaki et al., 1997).
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Hohenadl, C., Mann, K., Mayer, U., Timpl, R., Paulsson, M., & Aeschlimann, D. (1995). Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification. The Journal of Biological Chemistry, 270(40), 23415–23420. Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., & Engel, J. (1996). Structure of a novel extracellular Ca(2 +)-binding module in BM-40. Nature Structural Biology, 3(1), 67–73. Hohenester, E., Maurer, P., & Timpl, R. (1997). Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40. The EMBO Journal, 16(13), 3778–3786. Hohenester, E., Sasaki, T., Giudici, C., Farndale, R. W., & Bachinger, H. P. (2008). Structural basis of sequence-specific collagen recognition by SPARC. Proceedings of the National Academy of Sciences of the United States of America, 105(47), 18273–18277. Hughes, R. C., Taylor, A., Sage, H., & Hogan, B. L. (1987). Distinct patterns of glycosylation of colligin, a collagen-binding glycoprotein, and SPARC (osteonectin), a secreted Ca2 +-binding glycoprotein. Evidence for the localisation of colligin in the endoplasmic reticulum. European Journal of Biochemistry, 163(1), 57–65. Kelm, R. J., Jr., & Mann, K. G. (1991). The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation. The Journal of Biological Chemistry, 266(15), 9632–9639. Mason, I. J., Taylor, A., Williams, J. G., Sage, H., & Hogan, B. L. (1986). Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell ‘culture shock’ glycoprotein of Mr 43,000. The EMBO Journal, 5(7), 1465–1472. Mendoza-Londono, R., Fahiminiya, S., Majewski, J., Tetreault, M., Nadaf, J., Kannu, P., et al. (2015). Recessive osteogenesis imperfecta caused by missense mutations in SPARC. American Journal of Human Genetics, 96(6), 979–985. https://doi.org/10.1016/j. ajhg.2015.04.021. Nischt, R., Pottgiesser, J., Krieg, T., Mayer, U., Aumailley, M., & Timpl, R. (1991). Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40. European Journal of Biochemistry, 200(2), 529–536. Rosset, E. M., & Bradshaw, A. D. (2016). SPARC/osteonectin in mineralized tissue. Matrix Biology, 52–54, 78–87. https://doi.org/10.1016/j.matbio.2016.02.001. Sage, H., Vernon, R. B., Funk, S. E., Everitt, E. A., & Angello, J. (1989). SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca +2-dependent binding to the extracellular matrix. The Journal of Cell Biology, 109(1), 341–356. Sasaki, T., Gohring, W., Mann, K., Maurer, P., Hohenester, E., Knauper, V., et al. (1997). Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens. The Journal of Biological Chemistry, 272(14), 9237–9243. Sasaki, T., Hohenester, E., Gohring, W., & Timpl, R. (1998). Crystal structure and mapping by site-directed mutagenesis of the collagen-binding epitope of an activated form of BM-40/ SPARC/osteonectin. The EMBO Journal, 17(6), 1625–1634. Schneider, E. L., Thomas, J. G., Bassuk, J. A., Sage, E. H., & Baneyx, F. (1997). Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli. Nature Biotechnology, 15(6), 581–585. https://doi.org/10.1038/nbt0697-581.
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Trombetta-Esilva, J., & Bradshaw, A. D. (2012). The function of SPARC as a mediator of fibrosis. The Open Rheumatology Journal, 6, 146–155. https://doi.org/10.2174/ 1874312901206010146. Yost, J. C., Bell, A., Seale, R., & Sage, E. H. (1994). Purification of biologically active SPARC expressed in Saccharomyces cerevisiae. Archives of Biochemistry and Biophysics, 314(1), 50–63.
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