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 protei n purification on Zn(II)-IDA columns. These tags were fused to the green fl uorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA. Each fusio n protein exhibited elution heterogeneity (elution in two distinct pHs) fro m Zn(II)-IDA columns This led us to believe that two populations of fluores cent proteins were being expressed: one without the tag coeluting with Esch erichia coli proteins at pH 7.5 and one bearing the tap eluting at a pH low er 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 rGFPu v sequence which might be used as a secondary translation start site. This hypothesis was confirmed by changing the second ATG (methionine) codon to a n ACG (threonine) codon. The protein produced from this new construct elute d in a single fraction from a Zn(II)-IDA column. Thus, vector irregularitie s (along with other possibilities) should be examined when searching for th e cause of elution heterogeneity of a target protein, (C) 1999 Academic Pre ss.