why cDNA does not encode active enzyme?

PN pnokelai at whoccr.oulu.fi_no_spam
Tue Sep 29 02:22:23 EST 1998


Hi there,
I have cloned a new cDNA which should encode an enzyme according to
clones obtained from other species. However, this cDNA does not produce
active enzyme under CMV-promoter in cultured cells in conditions used
for the cDNAs from other species. cDNA has been cloned using PCR based
on EST-clones and RACE-techniques. To exclude a possibility of PCR
mutations, I cloned independently another fragments and they had
identical sequence compared to the first clones. In addition, critical
amino acids conserved among this protein superfamily (and needed in
catalysis) are present. The cDNA has 24 bp of 5’-non-coding and 90 bp of
3’-non coding regions. An Alu sequence starts 25 bp before assumed stop
codon. I shortened the UTRs without any effect.
What could be reasons for inactivity?

Pasi Nokelainen
Finland




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