Reconstitution of the peridinin-chlorophyll a-protein (PCP) from heterologously expressed apoprotein and isolated pigments

Abstract

DNA coding for the mature apoprotein of PCP was cloned into the expression vector pNDE. Constructs corresponding to the full length, and N-terminal domain only of the mainform PCP and full length, N and C terminal domains of the high salt HSPCP were expressed in E. coli. PCP apoproteins were induced by heat shock and extracted from washed inclusion bodies by buffer at 80o. Total pigments and lipids were extracted into 2-butanol and dried. For reconstitution, pigments were dissolved in ethanol and added to PCP apoproteins at 1:1 stoichiometry at a final ethanol concentration of 15% and held at 4o. Reconstitution was assessed by fluorescence emission at 673nm (478nm excitation) following 20x dilution with buffer. Reconstitution was successful with all constructs but differed quantitatively. N-domain alone PCP gave up to 25 % total incorporation of added pigments but required up to 48 hours incubation. Full length constructs were much quicker but the yield was lower. Purification by gel filtration gave reconstituted PCP, which was essentially identical to native PCP in absorbance, fluorescence and CD. Chl b, Chl d and acetyl Chl a could substitute for Chl a but not bacterioChl a or Chl c. Fucoxanthin and siphonaxanthin could not replace peridinin. Including Chl b or Chl d together with Chl a resulted in N-domain reconstructions showing energy transfer between the Chls. This suggests the single domain refolds and then forms dimers or piments can exchange after reconstitution.