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 cyan obacterium 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 v ivo with appropriate wild-type subunits to give constructs with the appropr iate oligomerization state and normal posttranslational modifications and w ith spectroscopic properties very similar to those of unmodified phycocyani n. All of these constructs were incorporated in vivo into the rod substruct ures of the light-harvesting complex, the phycobilisome. The C-terminal 114 -residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protei n (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coil and 40% in wild-type Anabaena sp. His-tagged phycocyanin b eta -BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. I n such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avid in-coated beads. Thus, the methods described here achieve in vivo productio n of stable oligomeric phycobiliprotein constructs equipped with affinity p urification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation, (C) 2001 Academic Press.