Expression and one-step purification of a developmentally regulated protein from Dictyostelium discoideum.
To overexpress Dictyostelium 5NT, a 1506bp fragment of the cDNA encoding the gene was cloned into a pET32c+ vector and expressed in the Escherichia coli expression host BL21-CodonPlus(DE3)-RIL by Isopropyl-beta-D-thiogalactoside (IPTG) induction. Maximum induction of insoluble recombinant protein was reached after incubation of the culture for 3h with 1.0mM IPTG. High level of 5NT expression was confirmed by SDS-PAGE and immunoblotting analysis. The recombinant 5NT was purified to homogeneity by a one-step purification using continuous-elution electrophoresis. Ten mg recombinant 5NT was purified per liter of growth medium. To achieve one of the goals of this study, polyclonal antibody against the recombinant 5NT was produced in a rabbit. We have shown previously by Northern blot and reporter gene analyses that 5nt is developmentally regulated. In this report, we used polyclonal antibody against the recombinant protein in Western blot analysis of membrane protein extracts from different developmental stages of Dictyostelium. The 5NT protein levels were first detected at the tight aggregation stage of development. Thus, there is no significant delay between transcription and translation of 5nt.
Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA.
Muatasem
M
Ubeidat
Charles L
CL
Rutherford
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004023:Dictyostelium / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D015800:Protozoan Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D000242:Cyclic AMP