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ARS Home » Research » Publications at this Location » Publication #79627
Title: MODIFIED RUBISCO ACTIVASE PROTEINS FOR ANALYSIS OF RUBISCO-RUBISCO ACTIVASE INTERACTION BY SURFACE PLASMON RESONANCE

Author
item Bertoni, Gregory
item PORTIS JR, ARCHIE

Submitted to: Plant Physiology Supplement
Publication Type: Abstract Only
Publication Acceptance Date: 8/6/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: We are exploring the feasibility of using surface plasmon resonance (SPR) technology to study the protein-protein interaction between Rubisco activase (RCA) and Rubisco. In SPR, one molecule is immobilized on a sensor chip and the binding of a second molecule presented in solution can be monitored in real time. Binding partners can be protein-protein, antibody-antigen, receptor-ligand, DNA-DNA, etc. To immobilize RCA without blocking any potential Rubisco binding site, we decided to add short tags to the N-terminus of RCA in order to bind RCA to the chip via the tag, which should expose native RCA to the solution containing Rubisco. We have created vectors to easily fuse either a T7 epitope tag or a hexahistidine tag (plus a short alanine spacer region) to the start codon of mature spinach activase. We purified T7-tagged RCA (T7-RCA) by ammonium sulfate precipitation and FPLC and showed it to have the same activity as untagged RCA in stimulating CO2 fixation by Rubisco. Using a SPR biosensor, we demonstrated covalent binding of an anti-T7-tag antibody to the sensor hip. We then showed that this immobilized "capture antibody" effectively and specifically binds tagged RCA. We are optimizing conditions to maximize the specificity of T7-RCA binding and to enable subsequent removal of the capture T7-RCA for chip regeneration and reuse to insure reproducibility. Our goals are to analyze the roles of activase, salt, and nucleotide concentration on activase aggregation and the kinetics of Rubisco-RCA binding and to study the role of specific activase protein sequences in this protein-protein interaction. This research was supported, in part, by Dept. of Energy grant #DE-A102-94ER20154.