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- Title
Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, nonpegylated and pegylated prolactin and prolactin receptor antagonist.
- Authors
Ocłon, Ewa; Solomon, Gili; Hayouka, Zvi; Salame, Tomer Meir; Goffin, Vincent; Gertler, Arieh
- Abstract
To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for largescale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under nondenaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.
- Subjects
PROLACTIN receptors; SURFACE plasmon resonance; CHEMICAL reagents; GENETIC mutation; GEL permeation chromatography
- Publication
PEDS: Protein Engineering, Design & Selection, 2018, Vol 31, Issue 1, p7
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzx062