+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist

Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist

Protein Engineering Design and Selection 31(1): 7-16

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale 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 non-denaturing 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.

Please choose payment method:

(PDF emailed within 0-6 h: $19.90)

Accession: 065361930

Download citation: RISBibTeXText

PMID: 29281090

DOI: 10.1093/protein/gzx062

Related references

Crystal structure of a prolactin receptor antagonist bound to the extracellular domain of the prolactin receptor. Journal of Biological Chemistry 283(27): 19085-19094, 2008

New reagents for poultry research: preparation, purification, and in vitro evaluation of non-PEGylated and mono-PEGylated chicken prolactin. Poultry Science 97(9): 3277-3285, 2018

Expression of the extracellular domain of the rat liver prolactin receptor and its interaction with ovine prolactin. Journal of Biological Chemistry 268(30): 22347-22352, 1993

Identification of JAK protein tyrosine kinases as signaling molecules for prolactin: Functional analysis of prolactin receptor and prolactin: Erythropoietin receptor chimera expressed in lymphoid cells. EMBO (European Molecular Biology Organization) Journal 13(11): 2583-2591, 1994

Identification of Jak protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells. The Embo Journal 13(11): 2583-2591, 1994

Endogenous human prolactin and not exogenous human prolactin induces estrogen receptor alpha and prolactin receptor expression and increases estrogen responsiveness in breast cancer cells. Journal of Steroid Biochemistry and Molecular Biology 88(1): 69-77, 2004

Preparation and characterization of recombinant prolactin receptor extracellular domain from rat. Molecular and Cellular Endocrinology 115(1): 1-11, 1995

Stimulatory effects of prolactin and anti prolactin receptor serum on prolactin binding sites in rat liver cells in suspension culture. Biochemical and Biophysical Research Communications 106(1): 243-249, 1982

Prolactin implants in the hypothalamus inhibit prolactin surges during pregnancy and alter prolactin release in response to dopamine receptor blockade. Journal of Neuroendocrinology 3(3): 249-252, 1991

Possible role of prolactin on bovine luteal function and the gene expression of prolactin and prolactin receptor in corpus luteum throughout the estrous cycle. Biology of Reproduction 68(Suppl. 1): 140, 2003

Prolactin causes the dissociation of prolactin from plasma membrane receptor in lactating mouse mammary cell: action of high prolactin concentration. Endocrine Journal 43(1): 93-100, 1996

Inhibitory effects of anti-prolactin receptor antibodies on prolactin binding in brain and prolactin-induced feeding behavior in ring doves. Neuroendocrinology 61(2): 125-135, 1995

Prolactin-binding proteins: studies on prolactin antibody and the prolactin receptor. Dissertation Abstracts International, B Sciences and Engineering 45(8): 2542, 1985

Identification of bovine prolactin in seminal fluid, and expression and localization of the prolactin receptor and prolactin-inducible protein in the testis and epididymis of bulls exposed to ergot alkaloids. Theriogenology 83(4): 662-669, 2015

Use of prolactin receptor antagonist to better understand prolactin regulation of pituitary homeostasis. Neuroendocrinology 98(3): 171-179, 2013