Retinal Transporter ABCA4 and Cloning of its Extracellular Domain ECD1

• Zyairr Bissoon, Biochemistry, University of Delaware
• Jazzlyn Jones, Applied Molecular Biology & Biotechnology, University of Delaware

• Esther Biswas, Medical and Molecular Sciences, University of Delaware

Z Bissoon1 , J Jones2, E Biswas-Fiss2

Department of Chemistry and Biochemistry, University of Delaware College of Arts and Sciences1, Department of Medical and Molecular Sciences, University of Delaware College of Health Sciences2, Newark DE 19716 

ABCA4 is part of a superfamily of ATP-binding cassette transporters. The ABCA4 protein is located in the retina and can be found in the outer segment of Retinal Transporter ABCA4 and Cloning of its Extracellular Domain ECD1. The rod and cone photoreceptors. As a transport protein it removes harmful and toxic retinoid compounds from the photoreceptors cells. ABCA4 has 3000 plus variants of its gene which can lead to a nonfunctional protein ultimately causing degenerative eye diseases such as Stargardt and Cone-rod dystrophy. Extracellular domain one (ECD1) is one of the four domains in the ABCA4 transporter. The sequence of ECD1 contains over six hundred amino acids and forms a loop-like structure that extends from the transmembrane domains of the protein. While the exact purpose of ECD1 is unknown, it is suspected that ECD1 plays a role in the structure of ABCA4 and retinal binding. Therefore ECD1 requires more research along with its variants to determine its role in the function of ABCA4. In this study, ECD1 was cloned using an E. coli expression system to gain more understanding on the domain and the ABCA4 protein. ECD1 was PCR amplified from a vector with the full length ABCA4 sequence. The ECD1 sequence was then ligated to a pET30 vector for E. coli  transformation and expression. After transformation, DNA from each clone was extracted and verified as recombinant by restriction digest. Analysis of the restriction digest confirmed that there were four recombinant ECD1 clones.  The cloning of the ECD1 gene will allow future expression and functional analysis (retinal binding) of the protein. This can provide a better understanding of ECD1’s role within ABCA4 and can permit characterization of patient variants in the ECD1 domain.