Production and purification of recombinant human SPARC

Methods Cell Biol. 2018:143:335-345. doi: 10.1016/bs.mcb.2017.08.020. Epub 2017 Dec 1.

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.

Keywords: BM-40; Baculovirus production; Extracellular matrix; Matricellular; Osteonectin.

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Cell Culture Techniques / instrumentation
  • Cell Culture Techniques / methods*
  • Cell Proliferation
  • Culture Media, Conditioned / chemistry
  • Extracellular Matrix / metabolism
  • Fibrillar Collagens / metabolism
  • Fibroblasts
  • Genetic Vectors / genetics
  • Osteonectin / isolation & purification*
  • Osteonectin / metabolism
  • Recombinant Proteins / isolation & purification*
  • Recombinant Proteins / metabolism
  • Sf9 Cells
  • Spodoptera

Substances

  • Culture Media, Conditioned
  • Fibrillar Collagens
  • Osteonectin
  • Recombinant Proteins
  • SPARC protein, human