Leptin tA Human, PEG

Leptin Antagonist Triple Mutant Human Recombinant, Pegylated
Cat. No.
BT22228
Source
Escherichia coli.
Synonyms
Appearance
White lyophilized (freeze-dried) powder.
Purity
Greater than 98.0% as determined by:
(a) Gel filtration analysis.
(b) Analysis by SDS-PAGE.
Usage
THE BioTek's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
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Description

Pegylated Leptin Antagonist Triple Mutant Human Recombinant is a single non-glycosilated polypeptide chain containing 146 amino and additional Ala at N-terminus acids and having a molecular weight of 35.6kDa, Leptin was mutated, resulting in L39A/D40A/F41A. However due to enlarged hydrodymanic volume it runs on the SDS-PAGE as 48 kDa protein and in gel-filtration on Superdex 200 as over 200 kDa protein. Leptin Antagonist Triple Mutant Human Recombinant is Mono-Pegylated with 20kDa PEG and was purified by proprietary chromatographic techniques.

Product Specs

Description
Pegylated Leptin Antagonist Triple Mutant Human Recombinant is a single, non-glycosylated polypeptide chain composed of 146 amino acids, with an additional alanine residue at the N-terminus. It has a molecular weight of 35.6 kDa. The Leptin sequence was mutated, resulting in the substitutions L39A/D40A/F41A. Due to its enlarged hydrodynamic volume, it appears as a 48 kDa protein on SDS-PAGE and elutes as an over 200 kDa protein in gel-filtration using Superdex 200. The Leptin Antagonist Triple Mutant Human Recombinant is mono-pegylated with a 20 kDa PEG molecule and was purified using proprietary chromatographic techniques.
Physical Appearance
White, lyophilized powder.
Formulation
The protein was lyophilized from a concentrated solution (0.65 mg/ml) containing 0.003 mM NaHCO3.
Solubility
It is recommended to reconstitute the lyophilized Leptin Antagonist Triple Mutant pegylated Human Recombinant in sterile 0.4% NaHCO3, adjusted to a pH of 8-9, to a concentration of at least 100 µg/ml. This solution can be further diluted with other aqueous solutions.
Stability
Lyophilized PEG-SHLA, though stable at room temperature for several weeks, should be stored desiccated below -20°C. After reconstitution at a concentration greater than 0.1 mg/ml and up to 2 mg/ml, filtered and sterilized mLEP mutant can be stored at 4°C or even room temperature for several weeks, making it suitable for long-term infusion studies using osmotic pumps. At lower concentrations, the addition of a carrier protein (0.1% HSA or BSA) is recommended. Avoid repeated freeze-thaw cycles.
Purity
Greater than 98.0% as determined by:
(a) Gel filtration analysis.
(b) SDS-PAGE analysis.
Biological Activity
This protein is capable of inhibiting the proliferation of BAF/3 cells stably transfected with the long form of the human leptin receptor when stimulated with leptin. Its in vitro activity is 6-8 fold lower than the non-pegylated antagonist. However, in vivo, it demonstrates a significant weight gain effect (compared to the non-pegylated antagonist), primarily due to increased food intake. Its in vivo activity is 9-27 fold higher compared to PEG-MLA.
Protein Content
Protein quantification was performed using UV spectroscopy at a wavelength of 280 nm. An absorbency value of 0.88 was used as the extinction coefficient for a 0.1% (1 mg/ml) solution at pH 8.0. This value was calculated using the PC GENE computer analysis program for protein sequences (IntelliGenetics).
Source
Escherichia coli.

Product Science Overview

Introduction

Leptin is a hormone predominantly made by adipose cells and enterocytes in the small intestine that helps to regulate energy balance by inhibiting hunger, which in turn diminishes fat storage in adipocytes. The Leptin Antagonist Triple Mutant (Human Recombinant, Pegylated) is a modified form of leptin designed to counteract the effects of endogenous leptin, making it a valuable tool in research related to obesity and metabolic disorders.

Structure and Composition

The Leptin Antagonist Triple Mutant is a single non-glycosylated polypeptide chain consisting of 146 amino acids with an additional alanine at the N-terminus. The molecular weight of this protein is approximately 35.6 kDa. However, due to its pegylation, it exhibits an enlarged hydrodynamic volume, causing it to run on SDS-PAGE as a 48 kDa protein and in gel-filtration on Superdex 200 as over 200 kDa protein .

Mutations

The antagonist properties of this leptin variant are achieved through specific mutations at positions L39A, D40A, and F41A. These mutations significantly reduce the binding affinity of leptin to its receptor, thereby inhibiting its biological activity .

Pegylation

Pegylation refers to the process of attaching polyethylene glycol (PEG) chains to a molecule, in this case, the leptin antagonist. The pegylation of the Leptin Antagonist Triple Mutant involves the attachment of a 20 kDa PEG molecule. This modification enhances the stability and solubility of the protein, prolongs its half-life in circulation, and reduces immunogenicity .

Production

The recombinant production of the Leptin Antagonist Triple Mutant is typically carried out in Escherichia coli. The protein is expressed, purified, and then pegylated using proprietary chromatographic techniques to ensure high purity and consistency. The final product is lyophilized from a concentrated solution containing 0.003 mM NaHCO3 .

Applications

The Leptin Antagonist Triple Mutant is primarily used in research to study the role of leptin in energy homeostasis, obesity, and related metabolic disorders. By inhibiting the action of endogenous leptin, researchers can better understand the physiological and pathological processes regulated by this hormone.

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