Cloning and Expression of the cbbO (0910) Gene from Halothiobacillus neapolitanus and Its Potential to Code for RubisCO Activase

Author

Emily F.E. Bustin, University of Southern Mississippi

Date of Award

Spring 5-2013

Degree Type

Honors College Thesis

Department

Chemistry and Biochemistry

First Advisor

Sabine Heinhorst

Advisor Department

Chemistry and Biochemistry

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase or RubisCO is an enzyme found in autotrophic organisms that functions to fix CO₂ and therefore plays an important role in the global carbon cycle. In order for RubisCO to increase its slow enzymatic rate, it must be exposed to high amounts of its substrate, CO₂. Carboxysomes, which sequester the RubisCO and its substrate, provide this function. It was previously believed that all the components necessary for carboxysome function were encoded by genes within the traditional cso operon. Recently, however, a gene in an operon located downstream of the cso operon was found to encode the novel shell protein CsoS1D. This discovery raised the possibility that other genes located outside of the traditional cso operon may contribute to the structure or function of the carboxysome. One such gene is cbbO, which encodes a potential RubisCO activase. A RubisCO activase could play a role in maintaining RubisCO’s catalytic efficiency within the carboxysome. To study the cbbO gene and its potential in greater detail, its gene first had to be over-expressed so that sufficient amounts of soluble recombinant CbbO protein could be purified for the generation of polyclonal antibodies. To achieve this goal, the cbbO gene of Halothiobacillus neapolitanus was PCR amplified and inserted into a plasmid vector. The genomic DNA was taken from the model organism for the study of carboxysomes, Halothiobacillus neapolitanus. The recombinant construct was sent for sequence determination. Then the cbbO gene fragment with the correct DNA sequence was ligated into a protein expression vector. Recombinant CbbO protein, which has a hexa-histidine affinity tag that is encoded on the pETDUET-1 vector, was purified by affinity chromatography and quantified and analyzed using SDS page. Finally, the recombinant CbbO protein was sent off for antibody generation in rabbits and initial bleeds were analyzed for antigen specificity using immunoblotting. The preliminary results seemed to indicate that an antibody probe for recombinant CbbO protein was obtained.

Copyright

Copyright for this thesis is owned by the author. It may be freely accessed by all users. However, any reuse or reproduction not covered by the exceptions of the Fair Use or Educational Use clauses of U.S. Copyright Law or without permission of the copyright holder may be a violation of federal law. Contact the administrator if you have additional questions.

Recommended Citation

Bustin, Emily F.E., "Cloning and Expression of the cbbO (0910) Gene from Halothiobacillus neapolitanus and Its Potential to Code for RubisCO Activase" (2013). Honors Theses. 156.
https://aquila.usm.edu/honors_theses/156