Date of Award
Doctor of Philosophy
Martin H. Spalding
Carbonic anhydrase (CA) catalyzes the reversible hydration of CO2. The periplasmic CA gene CAH1 of Chlamydomonas reinhardtii codes for a highly processed secreted glycoprotein. The primary translation product of the CAH1 gene is targeted to the algae's ER, where it is proteolytically processed to yield two different subunits, glycosylated, assembled into an active heterotetramer, and secreted. Expression of CAH1 in Escherichia coli and tobacco plants were effected in this study. For bacterial expression, two constructs, one with and another without, the algal target leader sequence were used. Similarly, after replacing the algal's target leader sequence with that from tobacco anionic peroxidase, expression of this gene in transgenic tobacco plants was also investigated;No expression was detected in bacteria from the construct with the algal leader. The gene was however efficiently expressed in the cytoplasm of E. coli from the construct without the leader sequence. The expressed protein which accumulated to very high levels, was confirmed to be periplasmic CA 1 (peri-CA1) based on the expected size and by cross-reaction with polyclonal antibody. The overexpressed polypeptide was present in inclusion bodies and no CA activity was detected in cell homogenates. The band was excised from polyacrylamide gel, and after some processing, used to raise polyclonal antibodies in rabbits;Of several transgenic plants screened by immunoblotting and found to express peri-CA1, a single plant, TL1, with a high level of expression was chosen for further analysis. SDS-PAGE gels of the purified protein from this tobacco plant, showed that it migrated as a series of discrete bands (two large and one small) with slightly faster mobility than the comparable bands in the purified algal protein. The expressed protein in the transgenic plant was active, and staining with thymol and sulfuric acid confirmed that it was also glycosylated. Analysis of vacuum infiltrates from leaves expressing peri-CA1 showed that it was enriched in the intercellular fluid. The sensitivity of the enzyme to sulfonamide inhibitors was similar to that of the native algal enzyme. These results suggest that the post translational processing of Chlamydomonas peri-CA1 is largely conserved in a higher plant.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Cyril Sebastian Roberts
Roberts, Cyril Sebastian, "Expression of Chlamydomonas reinhardtii periplasmic carbonic anhydrase in Escherichia coli and transgenic tobacco plants " (1994). Retrospective Theses and Dissertations. 11313.