Cloning and expression of human aldose reductase.

C Nishimura,D Carper,Y Matsuura,T Akera,T G Flynn,C Lyons,Y Kokai,N Morjana

doi:10.1016/s0021-9258(19)38740-x

Abstract

The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.

Highlights

Introduction of Aldose Reductase cDNA intoTransfer Vector-The aldose reductase cDNA fragment was excised by digestion with EcoRI from one of the muscle cDNA clones (L-l), and both ends were filled in with Klenow fragment
Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system
Our results indicate that this cDNA can be expressed as a functionally active enzyme using the baculovirus expression system and represents the structure of aldose reductase in the retina, the target organ for diabetic complications

Summary

Introduction

Introduction of Aldose Reductase cDNA intoTransfer Vector-The aldose reductase cDNA fragment was excised by digestion with EcoRI from one of the muscle cDNA clones (L-l), and both ends were filled in with Klenow fragment. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme.

Results

Conclusion