Vector engineering anomalies: impact on fusion protein purification performance

Recombinant protein purification using IMAC is often carried out by protein fusion to affinity tags. We have identified several tags useful for protein purification on Zn(II)-IDA columns. These tags were fused to the green fluorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA. Each fusion protein exhibited elution heterogeneity (elution in two distinct pHs) from Zn(II)-IDA columns This led us to believe that two populations of fluorescent proteins were being expressed: one without the tag coeluting with Escherichia coli proteins at pH 7.5 and one bearing the tag eluting at a pH lower than pH 7.5. Assessment of the constructs revealed the possibility of a ribosomal binding site and start codon between the fusion tag and the rGFPuv sequence which might be used as a secondary translation start site. This hypothesis was confirmed by changing the second ATG (methionine) codon to an ACG (threonine) codon. The protein produced from this new construct eluted in a single fraction from a Zn(II)-IDA column. Thus, vector irregularities (along with other possibilities) should be examined when searching for the cause of elution heterogeneity of a target protein.