A previously isolated partial cDNA sequence encoding human adenosine deaminase (ADA) was used to probe a cDNA library prepared from human cultured cell mRNA. Clones containing a combined overlapping length of 1462 bp were isolated and sequenced. One of these was found to include the entire ADA coding region An open reading frame consisting of 363 codons was identified, predicting a polypeptide of Mr 40 762. A mammalian expression plasmid was constructed, positioning the ADA coding sequence to be under transcriptional control of the mouse metallothionein promoter. Transfection of cultured mouse L-cells with this plasmid resulted in the acute expression of human ADA enzymatic activity, as assayed by isoelectric focusing.
Bibliographical noteFunding Information:
We areg ratefutl o Albert Tam and Michael Shepard for supplyingt he mousem etallothioneipnr o-moter,t o Chris Simonsenfo r usefuld iscussiona nd to Loes van der Voorn for ablet echnicaal ssistance. This work was supportedb y a grantt o D.V. fromt he NetherlandsO rganizationf or the Advancemenot f Pure Research( ZWO) and by USPHS grant no. AM 20428f rom the National Instituteso f Health.
Copyright 2014 Elsevier B.V., All rights reserved.
- Recombinant DNA
- eukaryotic expression vector
- metallothionein promoter