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P.N. Bishop, V.J. Hindson, T. Jowitt, P.G. Scott, M.M. LeGoff; The Dimerisation of Opticin in Solution . Invest. Ophthalmol. Vis. Sci. 2003;44(13):434.
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Purpose: Opticin is a glycoprotein that was initially identified bound to the collagen fibrils of the vitreous gel. It is a class III member of the extracellular matrix small leucine-rich repeat protein (SLRP) family and is highly expressed in the eye. In order to investigate its functions recombinant opticin was expressed and purified, but prior to undertaking these studies its biophysical properties were investigated. Methods: Bovine opticin was cloned and expressed in 293-EBNA cells using the pCEP-pu/AC7 vector. The recombinant protein was purified from conditioned media using ion-exchange and Jacalin lectin affinity chromatography. For comparison, opticin was extracted from vitreous collagen fibrils with guanidine hydrochloride, exchanged into non-chaotropic conditions and similarly purified. Both forms were subjected to CD spectroscopy and the recombinant form was analysed after gel filtration chromatography using multi-angle laser light scattering with a differential refractometer and by analytical ultracentrifugation. Results: Both the purified and extracted proteins showed similar profiles on SDS-PAGE analysis with opticin migrating at approximately 45 kDa. In addition, a minor 25 kDa opticin fragment was observed. The extracted and recombinant proteins showed similar CD profiles with ß-sheet and turn being the predominant identifiable secondary structures. Both the light-scattering and ultracentrifugation experiments demonstrated that the molecular weight of opticin in solution was 90,000-100,000. Conclusions: Opticin is a stable dimer in solution, but on denaturation behaves as a monomer. We have also shown that decorin, another SLRP family member, is a dimer in solution and this may be a general property of the SLRPs.
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