Cloning Of The Gene And Enzymatic Analysis Of NADP-dependent Retinol Dehydrogenase/reductase And Its Splicing Form In Human Liver

Huang,D. Y. etc purified and identified a novel NADP-dependent retinol dehydrogenase/reductase (NRDR) from rabbit liver cytosol in 1997. NRDR has much higher substrate specificity and affinity than reported alcohol dehydrogen-ase(ADH) and short-chain dehydrogenase/reductase (SDR) ,and its activity is widely spread in mammal liver, but in rabbit is the most. The full-length NRDR cDNA of rabbit and mouse is about 1200bp,and the full-length NRDR cDNA of human is about 1300bp. The size of their coding region is identical (783bp) , which can encode a protein of 260 amino acids. They had been cloned and submitted to GenBank ( [ AB045133 ] [ BC003484 ] [ AB045131 ]). However the activiy of NRDR in the human liver has not yet been studied on the molecular level. Here we report the whole process of the cloning of the NRDR cDNA and its short isoform form human liver tissue, the analysis of the gene structure and its expression, in order to discuss the biological activity and catalytic characterization of the NRDR and NRDR short isoform, that to show evidence for the study of the metabolic pathway of retinoids in liver.Methods1. cDNA cloning of NRDRTotal RNA was isolated, purified and identified from human and mouse liver, cDNA was acquired by reverse transcription, a pair of primers based on i-dentity sequence of the coding region of NRDR of human and mouse was used to amplify from the acquired cDNA by PCR, the PCR product was subcloned and analyzed. The full-length cDNA was amplified from human liver tissue by RACEmethod. According to TaKaRa 3' RACE kit . First , the Oligo dT-3sites Adaptor primer was used to conduct reverse transcription. Then, PCR using the primer F which was designed by sequence analyzing and primer R which was offered by the kit. The product was cloned and sequeneed. According to TaKaRa 5' RACE kit, first, reverse transcription was conduct by designing a 5' -end-phos-phorylated RT Primer based on the sequence analysis, then performed the degradation of RNA form hybrid DNA-RNA, and ligate the product to circularized form of single-stranded cDNA or formation of concatemers . Finally 5 ' -end region was acquired by nested PCR using two pairs of primers. PCR product was cloned and sequeneed. Sequence was analyzed by NCBI Blast, Jellyfish, Prosite and so on, and the molecular modeling was performed by GenoSD server, its result was viewed and analyzed by IMClite software. The distribution characterization of NRDR and sNRDR in fetal and adult was analyzed by RT-PCR and Northern blot.2. Expression of NRDR by E. coli Expression system The acquired cDNA was expressed in E. coli BL21-AI by using Gateway Expression system. First attB site was added to the coding region of NRDR by . modified primers, then the product was recombinated with the donor vector pDONR201 by BP recombination reaction to construct Gateway Entry clone containing NRDR coding region. Harvest and purify the entry clone and then recombinated with E. coli. expression vector by LR recombination reaction. pDEST17 containing NRDR coding region was constructed to obtain the expression of NRDR with a 6 x His-tag in the N-terminal. The pDESTl? containing NRDR coding region was transformed into E coli. BL21-AI, then the expression form and optimized exprssion condition were determined, and then expression it in large quantity. Inclusion body was isolated then dissolved by Guanidine HC1, and then refolded and purified by Ni2+ resin, the purified protein was eluted by imidazole gradient. The purifying efficiency was determined by HPSEC. Refolded protein was incubated with NADPH and retinal at 37^ , the produced retinol was detect by 5|xCI8 column to determine the catalytic activity of the purified protein, KM and Vmax of the catalytic reaction was determined by double reciprocal method.3. Expression and functional analysis of NRDR by mammal expression systemThe NRDR coding region was expressed in mammal cells by using the Gateway expressing system. First attE site was added to the coding region of NRDR by modified primers to acquire codin...