New reagents for poultry research: preparation, purification, and in vitro evaluation of non-PEGylated and mono-PEGylated chicken prolactin

被引:1
|
作者
Oclon, Ewa [1 ,2 ]
Solomon, Gili [1 ]
Hrabia, Anna [2 ]
Druyan, Shelly [3 ]
Hayouka, Zvi [1 ]
Gertler, Arieh [1 ]
机构
[1] Hebrew Univ Jerusalem, Inst Biochem Food Sci & Nutr, Robert H Smith Fac Agr Food & Environm, IL-76100 Rehovot, Israel
[2] Agr Univ Krakow, Dept Anim Physiol & Endocrinol, Al Mickiewicza 24-28, PL-30059 Krakow, Poland
[3] Volcani Ctr, ARO, Inst Anim Sci, Rishon Le Ziyyon, Israel
关键词
recombinant prolactin; PEGylation; pharmacokinetics; corticosterone; chicken; LARGE-SCALE PREPARATION; PROTEIN SECONDARY STRUCTURE; GROWTH-HORMONE; OVINE PROLACTIN; PLASMA; CORTICOSTEROIDOGENESIS; PROGESTERONE; ESTRADIOL; LEPTIN; PRL;
D O I
10.3382/ps/pey183
中图分类号
S8 [畜牧、 动物医学、狩猎、蚕、蜂];
学科分类号
0905 ;
摘要
Recombinant chicken prolactin (ch- PRL), expressed in Escherichia coli and purified as a monomer, was successfully PEGylated and purified to homogeneity as a mono-PEGylated protein (PEG-chPRL). Its biological activity was estimated by its ability to interact with human prolactin receptor extracellular domain (hPRLR-ECD) and stimulate PRLR-mediated proliferation in Nb2-11C cells. PEG-chPRL activity in a cell bioassay was 10-fold lower than that of non-PEGylated chPRL, but only 2-fold lower in a binding assay to hPRLR-ECD. The CD spectra of non-PEGylated and PEGylated chPRL were almost identical and similar to that of hPRL, indicating proper refolding. Although the PEGylation of chPRL resulted in lower activity in vitro, PEG-chPRL was absorbed more slowly than chPRL, remained in the circulation 16 h longer. Furthermore the effects of PEG-chPRL injections in chickens on subsequent corticosteroid levels in blood were significantly profound compared to chPRL. These favorable PEGylation-induced pharmacokinetic alterations should improve efficacy of PEG-chPRL in in vivo experiments, as dosing frequency can be reduced due to its prolonged persistence in the circulation, and thus reduce the frequency of dosing. Furthermore, hydrophobic interaction chromatography was successfully adopted to isolate PEG-chPRL as a better alternative for separation of PEGylated PRL, and is likely to be successfully applicable to other proteins.
引用
收藏
页码:3277 / 3285
页数:9
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