Human growth hormone-specific aptamer identification using improved oligonucleotide ligand evolution method

LETEG is a method developed and used for the separation and purification of proteins employing a single-step ligand (aptamers) evolution in which aptamers are eluted with an increasing temperature gradient. Using recombinant human growth hormone (rhGH) as the test purification target, and after avoi...

Full description

Saved in:
Bibliographic Details
Published in:Protein expression and purification 2010, Vol.69 (1), p.21-28
Main Authors: Calik, Pinar, Balci, Oğuz, Ozdamar, Tunçer H
Format: Article
Language:English
Subjects:
Aptamer
Aptamers, Nucleotide - analysis
Bacillus - metabolism
Bacterial Proteins - metabolism
Electrophoresis, Polyacrylamide Gel
Elution
Extracellular Space - metabolism
Fermentation
Human Growth Hormone - chemistry
Human Growth Hormone - isolation & purification
Humans
Hydrogen-Ion Concentration
Immobilized Proteins - isolation & purification
Kinetics
LETEG
Ligand
Ligands
MALDI-ToF
Protein
Purification
Recombinant Proteins - isolation & purification
rhGH
SELEX Aptamer Technique - methods
Separation
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
ssDNA
Temperature
Temperature gradient
Time Factors
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:LETEG is a method developed and used for the separation and purification of proteins employing a single-step ligand (aptamers) evolution in which aptamers are eluted with an increasing temperature gradient. Using recombinant human growth hormone (rhGH) as the test purification target, and after avoiding cross reactions of aptamers with Bacillus subtilis extracellular proteins by negative SELEX, the effects of time and pH on aptamer binding to rhGH were investigated. The highest binding efficiency of aptamers on rhGH-immobilized microparticles was obtained at pH 7.0. The aptamers that interacted with rhGH were eluted by a multi-stage step-up temperature gradient in Δ T = 10 °C increments within the range T = 55–95 °C; and the strongest affinity binding was disrupted at T = 85 °C where C Apt = 0.16 μM was eluted. The equilibrium binding data obtained was described by a Langmuir-type isotherm; where the affinity constant was K D = 218 nM rhGH. RhGH was separated from the fermentation broth with 99.8% purity, indicating that the method developed is properly applicable even for an anionic protein.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2009.05.015