Expression of recombinant human L-glutaminase in Escherichia coli: polyclonal antibodies production and immunological analysis of mouse tissues.
The first complete sequence of human L-glutaminase was deduced from breast cancer glutaminase cDNA cloned in our laboratory. This cDNA clone has now been engineered to synthesize both precursor and mature forms of the protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-3c) was found to be the most efficient for glutaminase overproduction. Upon induction, precursor glutaminase accounts for about 25% of total E. coli protein, whereas a lower amount (12%) was achieved for the putative mature protein. The optimal length of the translational spacer on the ribosome binding site was shown to be eight nucleotides. However, using this length of spacer, we were unable to obtain expression in the pQE vector, tagged with a 6x His sequence at the NH(2)-terminus, stressing the importance of the 5'-coding sequence in the expression efficiency. Although the precursor and mature recombinant forms of glutaminase were devoid of catalytic activity, the purified protein allowed us to obtain highly specific polyclonal antibodies, as shown by immunoblot analysis of mouse tissues. Furthermore, the antibodies were able to immunoprecipitate the in vitro translated enzyme using a reticulocyte lysate system; these antibodies might be a valuable tool for studies on L-glutaminase expression in mammalian tissues.
Campos
José A
JA
Laboratorio de Qui;mica de Proteínas, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Spain.
Aledo
J Carlos
JC
Segura
Juan A
JA
Alonso
Francisco J
FJ
Gómez-Fabre
Pedro M
PM
Núñez de Castro
Ignacio
I
Márquez
Javier
J
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005260:Female; D005972:Glutaminase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009928:Organ Specificity; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D018076:DNA, Complementary; D011994:Recombinant Proteins; D005972:Glutaminase