A reporter system for prokaryotic and eukaryotic cells based on the thermostable lichenase from Clostridium thermocellum.
The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.
Piruzian
E S
ES
Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina St., 3 GSP-1, 119991 Moscow, Russia. eleopiru@vigg.ru
Goldenkova
I V
IV
Musiychuk
K A
KA
Kobets
N S
NS
Arman
I P
IP
Bobrysheva
I V
IV
Chekhuta
I A
IA
Glazkova
D
D
D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells; D015870:Gene Expression; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics*; D016716:PC12 Cells; D011387:Prokaryotic Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase