Expression in Bacillus subtilis of the glycosomal glyceraldehyde-phosphate dehydrogenase gene from Trypanosoma brucei.
The cloned T brucei GAPDH gene was inserted within the B subtilis GAPDH gene, carried by pUC18. Upon transformation of B subtilis by this plasmid, not able to replicate in this host, the whole plasmid was inserted in the resident chromosome, presumably by a single recombination event between homologous, chromosomal and plasmid-borne sequences. The heterologous gene was expressed, as revealed by immunological reaction with monoclonal antibodies, recognizing specifically T brucei GAPDH. T brucei GAPDH, having little or no enzyme activity, comprises about 1.56% of cellular proteins. Peptide mapping showed that a fusion of a 7.5-kDa peptide had occurred to the N-terminal part of T brucei GAPDH. This fused protein is presumably the N-terminal part of B subtilis GAPDH, in agreement with the construction of the integrative plasmid.
Rayyan
W
W
Unit of Molecular Genetics, University of Louvain Medical School, Brussels, Belgium.
Kuntz
D A
DA
Opperdoes
F R
FR
Hoet
P P
PP
D000818:Animals; D000911:Antibodies, Monoclonal; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D010449:Peptide Mapping; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D014169:Transformation, Bacterial; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases