Enzyme-linked immunosorbent assay-based selection and optimization of elution buffer for TAG72-affinity...

Area of Science:

• Biochemistry
• Protein Purification
• Analytical Chemistry

Background:

• Affinity chromatography is crucial for purifying recombinant proteins.
• Selecting optimal elution buffers is critical for maximizing yield and minimizing protein denaturation.
• Current methods for buffer screening can be time-consuming and inefficient.

Purpose of the Study:

• To develop and validate a novel Enzyme-Linked Immunosorbent Assay (ELISA)-elution assay for rapid screening of elution buffers.
• To identify a superior elution buffer for the purification of the fusion protein FV/TNF-alpha using TAG72-affinity chromatography.
• To compare the efficacy of the developed assay with conventional elution buffer selection methods.

Main Methods:

• Development of an ELISA-elution assay to assess buffer performance.
• Screening of diverse elution buffer systems, including those with extreme pH, denaturants, chaotropic ions, and polarity-reducing reagents.
• Purification of FV/TNF-alpha using TAG72-affinity chromatography with selected buffers.

Main Results:

• Ammonia solution (1 M, pH 11.5) was identified as the optimal elution buffer.
• This buffer effectively dissociated the FV/TNF-alpha/TAG72 complex with minimal protein denaturation.
• A 300-fold increase in purified FV/TNF-alpha yield was achieved compared to a standard glycine-based elution buffer.

Conclusions:

• The developed ELISA-elution assay is highly effective for selecting optimal elution buffers in affinity chromatography.
• Ammonia solution (1 M, pH 11.5) provides a significantly improved method for FV/TNF-alpha purification.
• This approach enhances protein purification efficiency and yield, offering a valuable tool for researchers.