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Head of Institute: Prof. Oren Froy

Administrative manager: Ms. Yael Fruchter

Office Address:
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Robert H. Smith Faculty of Agriculture, Food and Environment,
The Hebrew University of Jerusalem, 
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Publications

2019
Gertler, A. ; Solomon, G. Pegylated Human Leptin D23L Mutant - Preparation and biological activity in vitro and in vivo in male ob /ob mice. Endocrinology 2019, 160, 891-898. Publisher's VersionAbstract
Recombinant monomeric human leptin (hLEP) and its D23L mutant were prepared in Escherichia coli and pegylated at their N-terminus using 20-kDa methoxy pegylated (PEG)-propionylaldehyde. As determined by both SDS-PAGE and size-exclusion chromatography, the pegylated proteins consisted of >90% monopegylated and <10% double-pegylated species. Circular dichroism spectra showed that their secondary structure, characteristic of all four α-helix bundle cytokines, was not affected by either the D23L mutation or pegylation. Because of the D23L mutation, affinity for hLEP receptor increased 25- and 40-fold for the pegylated and nonpegylated mutant, respectively. However, whereas the proliferation-promoting activity in vitro of nonmutated and mutated nonpegylated hLEP was identical, that of the respective pegylated mutant was approximately sixfold higher compared with the pegylated nonmutated hLEP. This difference was also seen in vivo. Both pegylated hLEPs at all doses significantly decreased body weight and food consumption, as compared with the vehicle-treated control. Once-daily administration of pegylated hLEP D23L at doses of 0.1, 0.3, and 1 mg/kg for 14 consecutive days in ob/ob mice resulted in significantly decreased body weight and food consumption as compared with respective pegylated hLEP-treated animals, with the biggest difference observed at 0.1 mg/kg. Repeated administration of either pegylated hLEP D23L or pegylated hLEP significantly decreased blood glucose levels compared with the control before glucose challenge and after oral glucose tolerance test, but with no difference between the two treatments. The pegylated hLEP D23L mutant seems to be a more potent reagent suitable for in vivo studies than the pegylated nonmutated hLEP. © Copyright 2019 Endocrine Society.
Ramos-Nieves, J. M. ; Giesy, S. L. ; Schwark, W. S. ; Gertler, A. ; Boisclair, Y. R. Technical note: Effects of pegylation and route of administration on leptin kinetics in newborn lambs. Journal of Animal Science 2019, 97, 3768-3775. Publisher's VersionAbstract
Chronic energy insufficiency resulting from inadequate feed intake or increased nutrient demand reduces plasma leptin in ruminants. Treatment of energy-deficient ruminants with exogenous leptin has identified some physiological consequences of reduced plasma leptin, but their full complement remains unknown. Additional leptin-dependent responses could be identified by using strategies that interfere with leptin signaling such as administration of leptin mutants that act as competitive antagonists. The effectiveness of these antagonists depends on their fold excess over endogenous leptin, and this condition can be achieved under in vivo conditions by extending the half-life (t1/2) of the antagonist by addition of a polyethylene glycol (PEG) molecule (pegylation). Use of this approach in ruminants, however, is limited by the lack of information on the t1/2 of native and pegylated leptin and on the most effective route of administration. To answer these questions, newborn lambs (n = 3) were injected with an intravenous (i.v.) bolus of 150 µg of human leptin followed by blood sampling over the next 12 h. Analysis of the semilog plasma leptin concentration over time yielded a t1/2 of 43 ± 4.9 min; an i.v. bolus of 276 µg of bovine leptin yielded a comparable t1/2 (P > 0.05). Next, newborn lambs (n = 4) received a single dose of 229 μg/ kg of metabolic body weight (BW0.75) of pegylated super human leptin antagonist (PEG-SHLA) via the i.v. or subcutaneous (s.c.) route. Plasma PEG-SHLA concentration reached a peak of 1,528 ± 78 ng/mL after 1 min and a nadir of 71 ± 9 ng/mL after 24 h with the i.v. route versus a peak of 423 ± 43 ng/mL after 300 min and a nadir of 146 ± 22 ng/mL after 24 h for the s.c. route; the t1/2 of PEG-SHLA was 394 ± 29 min for the i.v. route and 433 ± 58 min for the s.c. route. Finally, plasma concentration of PEG-SHLA was modeled when given either i.v. or s.c. at a dose of 229 μg/kg BW0.75 every 12 h. Once a steady state was reached, peak and lowest concentrations PEG-SHLA over the 12-h windows were 2,269 and 403 ng/mL for the i.v. route and 814 and 555 ng/mL for the s.c. route. Weighted PEG-SHLA concentrations over the 12-h period were 1,455 and 713 ng/mL for the i.v. and s.c. route, translating into 364- and 178-fold excess over endogenous plasma leptin. These data confirm the effectiveness of pegylation in extending the t1/2 of leptin antagonists in newborn lambs and in increasing their circulation in fold excess over endogenous leptin. © The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved.
2018
Ocłoń, E. ; Solomon, G. ; Hrabia, A. ; Druyan, S. ; Hayouka, Z. ; Gertler, A. New reagents for poultry research: preparation, purification, and in vitro evaluation of non-PEGylated and mono-PEGylated chicken prolactin. Poultry Science 2018, 97, 3277 - 3285. Publisher's VersionAbstract
ABSTRACT Recombinant chicken prolactin (chPRL), 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.
Oclon, E. ; Solomon, G. ; Hayouka, Z. ; Salame, T. M. ; Goffin, V. ; Gertler, A. 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 Eng Des Sel 2018, 31, 7-16.Abstract
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.
Ocłoń, E. ; Solomon, G. ; Hayouka, Z. ; Gertler, A. Preparation of biologically active monomeric recombinant zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) recombinant growth hormones. Fish Physiology and Biochemistry 2018, 44, 1215 - 1222. Publisher's VersionAbstract
Fish growth hormones (GHs) play an important role in regulating growth, metabolism, reproduction, osmoregulation, and immunity and have thus garnered attention for their application in aquaculture. Zebrafish GH (zGH) cDNA or rainbow trout GH (rtGH) cDNA was cloned into the pMon3401 vector, expressed in MON105-competent Escherichia coli and purified to homogeneity. Their biological activity was evidenced by their ability to interact with ovine GH receptor extracellular domain and stimulate GH receptor-mediated proliferation in FDC-P1-3B9 cells stably transfected with rabbit GH receptor. The relative affinity of zGH and rtGH, estimated by IC50, was about 38-fold and 512-fold lower, respectively, than ovine GH. This is likely the reason for the low biological activity in cells with rabbit GH receptor, ~ 36-fold lower for zGH and ~ 107-fold lower for rtGH than for human GH. This was not due to improper refolding, as evidenced by circular dichroism analysis. Predicting the activity of fish GHs is problematic as there is no one single optimal in vitro bioassay; heterologous assays may be ambiguous, and only homologous assays are suitable for measuring activity.