Single-step purification of recombinant human growth hormone (hGH) directly from bacterial osmotic shock fluids, for the purpose of (125)I-hGH preparation.
A good quality tracer, to be used in the radioimmunoassay of human growth hormone, was prepared by applying the chloramine-T iodination technique to the recombinant product obtained after a single-step high-performance size-exclusion chromatography purification of a bacterial osmotic shock fluid. The labeling reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of approximately 50% (NIDDK reference antiserum diluted 1:600,000). These values are very similar to those obtained by radioiodinating highly purified clinical-grade recombinant human growth hormone obtained from the same periplasmic extract after the regular six-step purification process. Both tracers provided the same accuracy, when evaluated with the use of commercial-quality control samples in a classical radioimmunoassay methodology, their stability being practically identical: about 18% decrease in antibody binding after 2 months of storage at -20 degrees C. The novel approach permits the utilization of transformed Escherichia coli strains as a source of freshly prepared, radioiodination-grade recombinant proteins, capable of providing better reproducibility and reagent continuity.
Teresa
M
M
Department of Application of Nuclear Techniques in Biological Sciences, IPEN-CNEN, São Paulo, 05508, Brazil.
Ribela
C P
CP
Camargo
I M
IM
Oliveira
J E
JE
Bartolini
P
P
D002700:Chloramines; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D009997:Osmotic Pressure; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014105:Tosyl Compounds
D002700:Chloramines; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D014105:Tosyl Compounds; C016300:chloramine-T; D019382:Human Growth Hormone