Vector engineering anomalies: impact on fusion protein purification performance.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Recombinant protein purification commonly employs affinity tags and Immobilized Metal Affinity Chromatography (IMAC).
• Zn(II)-IDA columns are a specific type of IMAC used for protein purification.
• Elution heterogeneity can complicate the analysis and purification of tagged proteins.

Purpose of the Study:

• To identify and evaluate affinity tags for protein purification on Zn(II)-IDA columns.
• To investigate the cause of observed elution heterogeneity in fusion proteins.
• To optimize recombinant protein expression and purification protocols.

Main Methods:

• Fusion of identified tags to recombinant green fluorescent protein (rGFPuv) using the pGFPuv vector.
• Analysis of fusion protein purification on Zn(II)-IDA columns.
• Genetic modification of the construct to alter a potential secondary translation start site (ATG to ACG).

Main Results:

• Fusion proteins exhibited elution heterogeneity, eluting at two distinct pHs from Zn(II)-IDA columns.
• Assessment of constructs suggested a secondary translation start site within the vector sequence.
• Modifying the secondary start codon eliminated elution heterogeneity, resulting in a single elution fraction.

Conclusions:

• Vector irregularities, such as internal ribosomal binding sites or start codons, can lead to expression of truncated or non-tagged proteins.
• These irregularities are a potential cause of elution heterogeneity in affinity tag-based protein purification.
• Careful examination of vector sequences and construct design is crucial for successful recombinant protein purification.