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2004
LSU-HHMI Summer Undergraduate Research Program |
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 Lacey
R. Howard, James V. Moroney Biological Sciences
Identification, Cloning, and Characterization of Cah8 in
Chlamydomonas reinhardtii
Carbonic anhydrases (CAs) are zinc-containing metalloenzymes
that are involved in important biochemical reactions such as
photosynthesis and respiration. They are a widely expressed
family of enzymes that catalyze the reversible hydration of
CO2. The roles played by CAs are an important aspect of the
CO2 concentrating mechanism (CCM) present in aquatic photosynthetic
organisms. The CCM enables these organisms to overcome the slow
diffusion of CO2 in water and thereby photosynthesize efficiently.
So far, eight CAs have been identified in Chlamydomonas
reinhardtii, a unicellular photosynthetic alga known to
have a CCM. In this study, we identified the eighth gene encoding
a ß-CA and designated this gene as Cah8. Nucleotide sequence
data show that Cah8 cDNA encodes an open reading frame with
a polypeptide of 333 amino acids and a theoretical molecular
mass of 35.8 kD. It is predicted to be targeted either to the
cytosol or chloroplast stroma. We fused the Cah8 DNA to the
coding sequence of the maltose binding protein (MBP) in a pMal
expression vector. Cah8 was overexpressed in Escherichia
coli then purified by amylose affinity column chromatography.
Determination of CA activity in an activity assay demonstrated
that Cah8 encodes an active CA. The purified recombinant fusion
protein was cleaved with Factor Xa to separate Cah8 from the
MBP and the isolated Cah8 is currently being used to raise an
antibody.
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