Researcher(s)
- Zyairr Bissoon, Biochemistry, University of Delaware
Faculty Mentor(s)
- Esther Biswas-Fiss, Medical and Molecular Sciences, University of Delaware
Abstract
Introduction: 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 the rod and
cone photoreceptors. As a transport protein it removes harmful and toxic retinoid
compounds from the photoreceptor cells. ABCA4 has 4000 plus variants in its gene
which can lead to a nonfunctional protein ultimately causing degenerative eye diseases
such as Stargardt and Cone-rod dystrophy. Extracellular domains, ECD1 and ECD2 are two of
six domains in the ABCA4 transporter. ECD1 and ECD2 are flexible regions of ABCA4 and form loop-like structures that extend from the transmembrane domains of the protein. While the exact purpose of ECD1 and ECD2 is unknown, it is suspected that both ECDs play a role in the structure of ABCA4 and retinal binding. Recent cryo-EM structures have also alluded to the ECDs role in retinoid interaction however more extensive research is needed to determine the impact of these domains on the overall function of ABCA4.
Approach: In this study, ECD1 and ECD2 were cloned according to the amino acid locations on the ABCA4 cryo-EM structures. ECD1 and ECD2 were PCR amplified from a vector with the full length ABCA4 sequence. The ECD1 and ECD2 sequences were then ligated to a pET28 vector for E. coli transformation and expression.
Results: After transformation, DNA from each clone was extracted and digested by restriction enzymes. Restriction digest analysis confirmed the presence of recombinant ECD1 and ECD2 clones.
Conclusion: The cloning of the ECD1 and ECD2 genes will allow future expression and functional analysis (retinal binding) of the proteins. This can provide a better understanding of the ECDs role within ABCA4 and can permit characterization of patient variants in the ECD1 and ECD2 domains.