Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli.
Recombinant mouse tryptophan hydroxylase (TPH) was expressed in large quantities in Escherichia coli strain MC 1061, using a bacterial expression vector, pKS, containing the full coding region of mouse TPH. Specific polyclonal antiserum to the subunit of the recombinant mouse TPH was produced in rabbit by injecting the TPH band cut from SDS-polyacrylamide slab gels. The resultant antiserum recognized a single identical protein band (MW = 54,000) from rat dorsal raphe area, pineal gland, and brain stem by Western blot analysis. The specific activity of recombinant mouse TPH obtained was equivalent to that of TPH purified from rat brain. The recombinant mouse TPH was stable for 3 days at 4 degrees C but lost 25% of the original activity for the same period at -20 degrees C. A serotonin concentration greater than 1 mM inhibited TPH activity under our assay conditions in a concentration-dependent fashion. The recombinant mouse TPH exhibited a charge isozyme corresponding to that of pineal gland TPH as applied to chromatofocusing column chromatography. Taken together, our results show that recombinant mouse TPH, expressed in large quantities in E. coli is not only enzymatically highly active but also shares many biochemical and immunochemical properties with native TPH.
Park
D H
DH
Laboratory of Molecular Neurobiology, Cornell University Medical College, Burke Medical Research Institute, White Plains, New York 10605.
Stone
D M
DM
Kim
K S
KS
Joh
T H
TH
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D051379:Mice / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014365:Tryptophan Hydroxylase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D014365:Tryptophan Hydroxylase