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 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 DeltaT=10 degrees C increments within the range T=55-95 degrees C; and the strongest affinity binding was disrupted at T=85 degrees C where C(Apt)=0.16muM was eluted. The equilibrium binding data obtained was described by a Langmuir-type isotherm; where the affinity constant was K(D)=218nM 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.
Calik
Pinar
P
Chemical Engineering Department, Middle East Technical University, 06531 Ankara, Turkey. pcalik@metu.edu.tr
Balci
Oğuz
O
Ozdamar
Tunçer H
TH
D052157:Aptamers, Nucleotide / Q000032:analysis*; D001407:Bacillus / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000378:metabolism; D005285:Fermentation; D019382:Human Growth Hormone / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000302:isolation & purification; D007700:Kinetics; D008024:Ligands; D011994:Recombinant Proteins / Q000302:isolation & purification; D052156:SELEX Aptamer Technique / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors
D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D055767:Immobilized Proteins; D008024:Ligands; D011994:Recombinant Proteins; D019382:Human Growth Hormone