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

Area of Science:

• Biochemistry
• Biotechnology
• Molecular Biology

Background:

• Protein purification is crucial for biopharmaceutical development.
• Existing methods can be complex and costly.
• Aptamers offer specific molecular recognition for purification applications.

Purpose of the Study:

• To develop and evaluate a single-step aptamer-based protein purification method.
• To optimize aptamer binding conditions for recombinant human growth hormone (rhGH).
• To assess the purity of rhGH purified using the novel method.

Main Methods:

• Developed a ligand (aptamer) evolution technique (LETEG) with temperature gradient elution.
• Utilized negative SELEX to eliminate cross-reactivity with bacterial proteins.
• Investigated the impact of pH and time on aptamer-rhGH binding.
• Characterized binding affinity using Langmuir isotherm and determined elution temperature.

Main Results:

• Optimal aptamer binding to rhGH occurred at pH 7.0.
• Aptamers were eluted using a multi-stage temperature gradient (55-95°C).
• Strongest binding disrupted at 85°C, eluting aptamers at 0.16µM.
• Purified rhGH achieved 99.8% purity.
• Binding affinity constant (K(D)) was 218nM for rhGH.

Conclusions:

• The LEVANT method provides efficient single-step purification of proteins using aptamers.
• The method is effective for purifying anionic proteins like rhGH.
• LETEG demonstrates high applicability and potential for industrial protein purification.