Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was p urified, and polyclonal antibodies were raised against this protein. The an tibodies were subsequently used to screen a cDNA library that was construct ed using RNA from 2nd instar larvae. Eight identical cDNA clones were isola ted. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana gluta thione S-transferase (CfGST). Identity of CfGST was confirmed by using affi nity-purification as well as enzyme activity assay. CfGST was closer in sim ilarity to insect GST2 members than GST1 members. The apparent V-max of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitro benezene (CDNB) were similar. However, the enzyme had a three-fold higher a ffinity towards CDNB than glutathione. Analyses using Northern blot, immuno blot and immunocytochemistry demonstrated that the fat body was the major t issue where the enzyme was synthesized and stored. Higher levels of CfGST p rotein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may ha ve other roles during insect development that are not readily apparent at p resent. The CfGST cDNA was expressed in a recombinant baculovirus expressio n system and an active enzyme was produced. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.