Date Approved
12-2015
Document Type
Thesis
Degree Name
Master of Science in Biomedical Sciences
Department
Cell Biology
College
Graduate School of Biomedical Sciences
Funder
Continuation of work previously supported by a grant from the National Institutes of Health, National Eye Institute (NIH-EY 013113 to E.E.B-F)
First Advisor
Natalia Shcherbik, PhD
Committee Member 1
John Pastorino, PhD
Committee Member 2
Gary Goldberg, PhD
Subject(s)
ATP Binding Cassette Transporter Subfamily A Member 4; Mammals; ABCA4 Transporter; Retinal Photoreceptor Cell Outer Segment
Disciplines
Cell Biology | Genetic Processes | Medical Cell Biology | Medicine and Health Sciences | Molecular Genetics
Abstract
Purpose- ABCA4 is an ATP Binding Cassette (ABC) protein localized at cone and rod photoreceptor outer segments of the retina. Though mutations in ABCA4 have been found to cause a broad spectrum of disorders, ABCA4's high molecular weight (~210 kDa) and biochemical transmembrane properties have made ABCA4 functional studies unsuccessful. Identifying how ABCA4 functions might have significant clinical impact and open unanticipated avenues for therapeutic interventions. Thus, the objective of this study was to develop a new expression system able to produce the full-length, correctly folded and fully functional ABCA4 protein.
Methods- the ABCA4 gene was amplified from the PCMV6-ABCA4 entry vector (available in laboratory), purified and cloned into the transfer vector, pACBacl, under the control of the polh, "very late" promoter. The recombinant construct, pACBac l-ABCA4, was transfected into DH10 E. coli cells containing both Bacmid DNA and VLP genes. Subsequently, the transformed DNA was purified and transfected into SF2 l insect cells. The Baculovirus both developed and propagated in the SF21 cell-line, expecting to produce both the ABCA4 protein and the VLPs simultaneously. Verification of ABCA4 expression in the SF2 l cell-line was achieved via a Western Blot analysis using ECO I/ECD2 domain specific antibodies.
Results- Our data does not show definitively that ABCA4 has produced inside the SF2l insect cell line, however, there is potential for this generation to occur. We have successfully cloned pACEBACI-ABCA4 into the Bacmid DNA, and a Western Blot analysis revealed a-210 kDa polypeptide that cross-reacted with domain specific ABCA4 antibodies ECD l/ECD2 with degradation. More studies are needed to determine optimized ABCA4 production in SF21. The next step will be to evaluate A BCA4 presence in the media, which will indicate efficiency of its packaging into VLP vesicles.
Recommended Citation
Ortiz, Amaryllis, "The Development of a Platform for Expression and Purification of the Mammalian Transmembrane Transporter ABCA4" (2015). Graduate School of Biomedical Sciences Theses and Dissertations. 70.
https://rdw.rowan.edu/gsbs_etd/70
Included in
Cell Biology Commons, Genetic Processes Commons, Medical Cell Biology Commons, Molecular Genetics Commons
