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Cloning, Expression and Characterization of Novel Aldo‐Keto Reductase 1B (AKR1B) Proteins ‐ Human AKR1B11 and Murine Akr1b16
Author(s) -
Salabei Joshua Kuimeta,
Bhatnagar Aruni,
Barski Oleg A
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.666.4
Subject(s) - open reading frame , microbiology and biotechnology , biology , complementary dna , gene , immunoprecipitation , western blot , exon , messenger rna , transfection , human genome , gene expression , peptide sequence , biochemistry , genome
The Aldo‐Keto reductase (AKR) superfamily comprises enzymes that catalyze redox transformations. A novel gene locus, tcag7.1260 ( AKR1B11 ) has been predicted to cluster with human members of the AKR1B subfamily, AKR1B1 and AKR1B10. AKR1B11 is predicted to have 10 exons and an open reading frame having 91% and 67% identities with AKR1B10 and AKR1B1 respectively. Cross genome comparison of human and murine AKRs reveals the existence of a murine AKR gene, 2310005E10Rik ( Akr1b16) , having an open reading frame with 82.9% identity with human AKR1B10 protein. We test the hypothesis that AKR1B11 and Akr1b16 are expressed into functional proteins. Using whole human/mouse tissue mRNA, we demonstrated the existence of spliced AKR1B11 and Akr1b16 mRNAs in human brain and testes, and in multiple murine tissues. cDNAs were cloned into pET28a and pIRES‐hrGFP‐1α vectors for bacterial and mammalian expression respectively. Both genes were expressed as 37 kDA proteins. COS 7 cells transfected with pIRES‐hr‐GFP‐1α/ AKR1B11 showed a 4.8 fold (with 500μM p‐NB) and 3.3 fold (with 10mM DL‐glyceraldehyde) increase in enzymatic activity. We raised specific antibody against AKR1B11. Immunoprecipitation and Western blot showed the presence of AKR1B11 protein in the ovary, testes and kidney. AKR1B11 protein was also detected in human ovary sections by immunohistochemistry. We conclude that AKR1B11 is present in human tissues. Further confirmation of its activity in these and other tissues is needed. Protein activity measurements with a broad variety of AKR substrates are required for the characterization of AKR1B11 and Akr1b16. Research support: COBRE: P20RR024489