Recombinant somatolactin as a stable and bioactive protein in a cell culture bioassay: development and validation of a sensitive and reproducible radioimmunoassay.
A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA. In contrast to native proteins, which tend to dimerize and aggregate immediately after pituitary isolation, the majority of recombinant sole SL (rsSL) remained as a monomeric protein after long-term storage, as shown by size exclusion chromatography and Western blot. Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED50) of the assay were 0.15 and 1.8-2.1 ng/ml respectively. Intra-and interassay coefficients of variation were 4.3% and 6.5% at ED50 levels. Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism between rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed. In order to demonstrate some biological activity of rsSL, the ability of this recombinant product to prime gilthead sea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay. A bell-shape dose-response curve was obtained with a maximum at 50 nM (1.2 micrograms/ml), similar to that reported previously for GH. Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which retains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.
Calduch-Giner
J A
JA
Instituto de Acuicultura de Torre de la Sal, CSIC, Castellón, Spain.
Pendón
C
C
Valdivia
M M
MM
Pérez-Sánchez
J
J
D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D029941:Fish Proteins; D005412:Flatfishes; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010902:Pituitary Gland / Q000737:chemistry; D010907:Pituitary Hormones / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011863:Radioimmunoassay; D011994:Recombinant Proteins
D029941:Fish Proteins; D006023:Glycoproteins; D010907:Pituitary Hormones; D011994:Recombinant Proteins; C064448:somatolactin protein, fish