Cloning and reconstitution of PORA from Arabidopsis thaliana

Abstract

Protochlorphyllide oxidoreductase, POR, is an enzyme found in the prolamellar bodies in etioplasts. Three isoforms have been detected so far; PORA, PORB and PORC. PORA is found in the etiolated seedlings, PORB is present at all times and PORC is thought to have a photoprotective role. POR is a light dependent enzyme that catalyzes the transformation from protochlorphyllide (pchlide) to chlorophyllide (chlide). This step is the only light regulated step in the biosynthesis of chlorophyll. In this thesis a mature product of PORA from Arabidopsis thaliana has been cloned into a pET151 vector. PCR sequencing has confirmed that the 1065 nucleotide long sequence is correct. The mature product of PORA (AU2), has been expressed by E. coli and purified by centrifugation, filtration and metal chelate affinity chromatography. Expected size of AU2 is 40kDa where 2 of the kDa is a histidine-tag. Mass spectrometry (MS) analysis confirmed that AU2 is PORA from Arabidopsis thaliana. SDS PAGE revealed several protein bands of the purified AU2. The band of highest molecular weight was PORA, the other lower band is probably AU2 of shorter length, or degraded AU2. Ratio between POR and pigments in extracted etioplasts from 4.5 day dark grown barleys were determined to be 2.7x106 etioplasts per 1ng of POR. Reconstitution with AU2 and pigments in that ratio with NADPH in excess were performed, but in the absorbance spectra AU2 did not convert pchlide into chlide. Future use of cloned POR products will be in reconstitution experiments. And possible protein-protein interactions to itself and Lil3 protein.