Expression, Purification, Activity Identification And Polyclonal Antibody Production Of Rabbit NADP(H)-Dependent Retinol Dehydrogenase/Reductase

Retinoids participate in a wide spectrum of physiological processes as a physiological active compound for many vertebrates, by means of gene regulation. These processes include morphogenesis, cell growth, differentiation, immunological function regulation, etc. In addition, it also shows extensive biological effects range from inhibition of tumor cell proliferation, induction of tumor cell apoptosis to anti-infection, etc. Retinoids homeostasis in vivo was influenced by various factors such as diets and the relevant enzymes. The mechanisms underlying the regulation of retinoids metabolism by relevant enzymes, however, are not fully demonstrated.NADP(H)-dependent retinol dehydrogenase/reductase (NRDR), an enzyme purified and identified in 1997 from rabbit liver cytosol by Huang, with high substrate affinity and specificity to retinal in vitro, is involved in the oxidoreduction process between retinol and retinal. The transformation between retinol and retinal is the rate limited step for retinoic acid synthesis in vivo. NRDR thus may play an important role in the modulating retinoids homeostasis. And the functional expression of NRDR is supposed to contribute to further research.NRDR cDNA was cloned from the rabbit liver and the coding region of NRDR was constructed to the Gateway-based expression vector pDESTTM 17, which was transformed into the E.coli BL21-AI to express the protein. Alter sonication and centrifugation of the bacteria, the soluble NRDR in supernatant was purified by affinity chromatography. Furthermore, the kinetic parameters of NRDR were determined by reverse phase high performance liquid chromatography (HPLC). The purified protein was used to immunize guinea pig to prepare polyclonal antibodies which were finally analyzed by Western-Blot.The soluble NRDR with high activity for reducing retinal to retinol was obtained, of which the optimal harvest time was tested to be 4 hours, with a 41.4% expression level. Moreover, the homogeneous enzyme was obtained by a single step affinity chromatography, with purity up to 97.2% and specific activity 64 fold enhanced. Furthermore, the kinetic parameters of NRDR were determined by HPLC. The values of the Km and the Vmax were (4.55±0.33)μmol/L and (0.307±0.065)μmol/(min·mg), respectively, which shows minor difference from that of native NRDR obtained from the rabbit liver. The anti-NRDR polyclonal antibodies performed well in Western-Blot assay for NRDR and were supposed to facilitate the research of NRDR.