The Expression and Characterization of Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Tobacco
Witt, William T.
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The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most studied membrane protein models because of its clear clinical significance. Mutations within the CFTR gene lead to cystic fibrosis, the most common autosomal recessive genetic disorder in the Caucasian population. CFTR, a large 160 kDa glycoprotein, is a chloride ion channel in the ABC superfamily of transporter proteins. Due to low natural abundance of CFTR and difficulties producing sufficient amounts in heterologous systems, the exact protein function/structure relationship is unknown. Expression of CFTR in E. coli is lethal and mammalian culture systems are expensive and low yielding. However, successful bioproduction of many complex human proteins has been shown in transgenic plants. Our research objective is to develop tobacco as a model system for expressing human CFTR. Constructs of full-length CFTR fused to the 35S double enhanced promoter could not be propagated in E. coli, suggesting that the CFTR product generated by â leakyâ expression was detrimental to bacteria. Two strategies were undertaken to address the problem: 1) a plant intron was introduced into CFTR sequence and 2) a more tightly regulated wound-inducible promoter MeGATM was used. Tobacco was transformed with all constructs. CFTR presence was determined by polymerase chain reaction (PCR). Expression and intron splicing was analyzed by reverse transcriptase-PCR. Splicing did not occur presumably due to intron /exon contexts. In tobacco expressing MeGA:CFTR, however, novel high-molecular-weight membrane-associated proteins were immunodetected using anti-CFTR antibodies suggesting that tobacco may be capable of producing human CFTR.
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