Overproduction and partial purification of the Norrie disease gene product, norrin, from a recombinant baculovirus.
Abnormal vascularization of the peripheral retina and retinal detachment are common clinical characteristics of Norrie disease (ND), familial exudative vitreoretinopathy, Coats' disease, and retinopathy of prematurity. Although little is known about the molecular basis of these diseases, studies have shown that all of these diseases are associated with mutations in the ND gene. In spite of this, little is known about norrin, its molecular mechanism of action, and its functional relationship with the development of abnormal retinal vasculature. To obtain a large quantity of norrin for structural and functional studies, we have overproduced it in insect cells. For this purpose, a cDNA fragment (869 bp) was isolated from a human retinal cDNA library by amplification and was cloned into an expression vector. The purified plasmid was co-transfected with wild-type linearized Bac-N-Blue DNA into S. frugiperda Sf21 insect cells. The recombinant virus plaques were purified and clones were selected based on the level of recombinant protein expressed in Sf21 cells infected with a purified recombinant virus. From these, a high-titer stock was generated and subsequently used to prepare a fused protein on a large scale. The protein was partially purified by the process of immobilized metal affinity chromatography and the use of ion exchange chromatography
Shastry
Barkur S
BS
Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA. shastry@oakland.edu
Trese
Michael T
MT
D000015:Abnormalities, Multiple / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism*; D001766:Blindness / Q000378:metabolism; D003001:Cloning, Molecular; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015792:Retinal Dysplasia / Q000378:metabolism; D013577:Syndrome; D014162:Transfection / Q000379:methods
D005136:Eye Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins