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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., (© 2018 Elsevier Inc. All rights reserved.)

SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40), a matrix-associated protein, disrupts cell adhesion and inhibits the proliferation of many cultured cells. We report the expression of recombinant human protein (rhSPARC) in a baculovirus expression system. This procedure routinely yields approximately 1 mg of purified protein per 500 ml of culture supernate. rhSPARC produced by insect cells migrates at the appropriate molecular weight under reducing and nonreducing conditions. The rhSPARC purified from insect cell media appeared structurally similar to SPARC purified from mammalian tissue culture by the criterion of circular dichroism. In addition, a series of anti-SPARC and anti-SPARC peptide antibodies recognized insect cell rhSPARC. We also show that rhSPARC produced in this system is glycosylated and is biologically active, as assessed by inhibition of endothelial cell proliferation and induction of collagen I mRNA in mesangial cells. Significant amounts of rhSPARC can now be generated in the absence of contaminating mammalian proteins for structure/function assays of SPARC activities., (Copyright 2000 Academic Press.)

Expression of SPARC (secreted protein acidic and rich in cysteine), a 43-kDa extracellular matrix-associated glycoprotein involved in tissue remodeling, was quantitated during normal human keratinocyte (NHK) growth in culture and as a function of sodium n-butyrate (NaB)-induced differentiation to mature enucleate cornified envelopes (CEs). Low levels of SPARC expression were observed in the basal-like cells of control NHKs, with isolated cells showing intense SPARC expression on the ventral surface. After addition of NaB, SPARC expression increased and the pattern of expression shifted to one involving predominantly suprabasal cells (i.e., spinous cells, pre-CEs, and mature CEs). Dense deposits of SPARC often surrounded the mature CEs. Flow cytometric analysis indicated that approximately 13% of NHKs expressed SPARC within 24 h of seeding into culture. This fraction of SPARC+ cells increased with time and peaked immediately postconfluence (31.3 +/- 6.3% SPARC+). Cellular SPARC expression then decreased to baseline levels during entrance into plateau phase growth. SPARC was detectable in all phases of the cell cycle. SPARC levels were more intense and heterogeneous within the G2/M and G1 phases while S phase cells exhibited relatively homogeneous, low intensity, SPARC expression. During NaB-induced NHK differentiation, SPARC intracellular content increased prior to the onset of CE formation (i.e., 2 days after its addition) followed by a period of extracellular accumulation which coincided with the time of maximal CE generation (i.e., Days 4 and 5 after NaB addition). Correlation of cell size with anti-SPARC immunoreactivity revealed a predominance of SPARC expression in cells with a suprabasal phenotype. NHKs cultured on fibronectin (FN), an established modulator of epidermal cell maturation in vitro, showed a similar response to NaB. In general, however, the level of NaB-induced SPARC expression was considerably reduced in FN cultures correlating with a lower efficiency of CE formation. Induced SPARC expression was, in large part, dependent on autocrine transforming growth factor-beta (TGF-beta) production since incubation in the presence of NaB+neutralizing antibodies to TGF-beta inhibited both the expression of SPARC by 72% and development of mature CEs.