Expression and characterization of the soluble form of recombinant mature HSV-2 glycoprotein G for use in anti-HSV-2 IgG serodiagnostic immunoassay.

Herpes simplex virus type-2 (HSV-2) specific glycoprotein G (gG-2) is widely used as the antigen of choice for serodiagnosis of HSV-2. In order to develop an ELISA for serodetection of HSV-2 IgG in patient sera, the soluble form of the mature gG-2 antigen (mgG-2), gG283-649, was expressed using a baculovirus expression system. gG283-649 contains the complete extracellular domain of mgG-2 including the C-terminal region, which despite homology to gG-1, does not cross-react with HSV-1 antibodies present in HSV-1 positive patient sera. gG283-649 had increased performance compared to a previously described gG-2 fragment and showed high sensitivity and specificity in a method comparison with HerpeSelect 1 & 2 Immunoblot IgG, a commercially available FDA-cleared assay for serodetection of HSV-1 and 2 antibodies. A total of 234 clinical samples consisting of 134 high risk samples, including 45 samples from pregnant subjects, and a panel of 100 mixed diagnosis samples, spanning the measurable range were tested in the method comparison. Clinical sensitivity and specificity were determined to be 94.2% and 100%, respectively. We conclude that this soluble form of mgG-2 is a novel antigen of choice for developing an ELISA for type-specific serodiagnosis of HSV-2.

The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II.

Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.