Retinoblastoma protein purification and transduction of retina and retinoblastoma cells using improved alphavirus vectors.
OBJECTIVE
To develop and use improved Semliki Forest vectors (SFVs) for functional and structural analyses of the retinoblastoma protein (RB) in developing retina and retinoblastoma cells.


METHODS
Virus was harvested from cells transfected with replicon and helper plasmids. Combinations of producer and target cells were tested for optimal virus production and protein expression. The replicon was improved by adding an expanded multiple cloning site, translational enhancer, and FLAG and HIS10 epitope and affinity tags. Affinity chromatography was used to purify beta-galactosidase or RB. RB function was assessed through interaction with E2F1. The efficacy of SFV as a retinal delivery system was tested on mouse explants and cultured human retinoblastoma cells.


RESULTS
The optimal producer and target cell lines were an HEK-293 derivative (Phoenix Eco) and BHK-21, respectively. Stable expression of structural proteins in the BHK-21 helper line simplified virus production and amplified virus yield 100-fold. The translational enhancer improved expression three- to eightfold. Full-length, functional RB was produced without the truncated products characteristic of bacterially produced RB and was purified using the affinity tags. SFVs efficiently transduced mouse retinal explants and multiple hard-to-transduce retinoblastoma tumor lines.


CONCLUSIONS
This study describes a simple, rapid, SFV vector system to produce recombinant proteins, such as RB, in mammalian cells. These SFV vectors represent an efficient approach to purification of proteins and protein complexes and transduction of retinal or retinoblastoma cells for the purpose of in vivo analysis of protein functions.
Ontario Cancer Institute Cancer Informatics, University Health Network, Toronto, Canada.
David P
DP
DiCiommo
Allison
A
Duckett
Irina
I
Burcescu
Rod
R
Bremner
Brenda L
BL
Gallie
D000524:Alphavirus / Q000235:genetics*; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D012093:Replicon; D012160:Retina / Q000378:metabolism*; D019572:Retinal Neoplasms / Q000378:metabolism / Q000473:pathology; D012175:Retinoblastoma / Q000378:metabolism* / Q000473:pathology; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000302:isolation & purification*; D014035:Togaviridae / Q000235:genetics / Q000254:growth & development; D014157:Transcription Factors / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D014764:Viral Proteins / Q000096:biosynthesis
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D014764:Viral Proteins; D004247:DNA