Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains.
A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.
Cai
Y A
YA
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
Murphy
J T
JT
Wedemayer
G J
GJ
Glazer
A N
AN
D000595:Amino Acid Sequence; D017033:Anabaena / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D045342:Light-Harvesting Protein Complexes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D045524:Phycobilisomes; D010798:Phycocyanin / Q000737:chemistry / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D045524:Phycobilisomes; D010940:Plant Proteins; D011994:Recombinant Proteins; D010798:Phycocyanin; D006639:Histidine