title: Vector engineering anomalies: impact on fusion protein purification performance.
abstract: 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.
authors.affiliation: Department of Chemical Engineering and Center for Biotechnology and Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Pasquinelli
authors.forename: R R
authors.initials: RR
authors.lastname: Koepsel
authors.forename: N
authors.initials: N
authors.lastname: Wu
authors.forename: M M
authors.initials: MM
authors.lastname: Ataai
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D006632:Histamine / Q000031:analogs & derivatives; D006639:Histidine / Q000235:genetics; D007093:Imidazoles; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D015032:Zinc
chemical_list: D007093:Imidazoles; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D006632:Histamine; C005954:imidazoleacetic acid; D015032:Zinc