Selection of optimum affinity tags from a phage-displayed peptide library. Application to immobilized copper(II) affinity chromatography.
Immobilized metal affinity chromatography (IMAC) is a versatile tool for the purification of proteins with affinity for immobilized metals. Moreover, this technique has also been used for the separation of proteins that do not exhibit significant metal affinity in the native form, by their fusion to a short metal-binding peptide (a tail), most commonly, a sequence consisting of six adjacent histidine residues (His6). A phage-displayed random hexamer library is used to select for peptides with affinity for immobilized copper. The study follows our previous investigation in which a stringent selection protocol led to the selection of only one copper-binding peptide containing two histidines. The less stringent conditions employed in this work resulted in the selection of a more diverse population of peptides, but again, dominated by peptides containing two histidines (13 out of 19). The prevalence of peptides with two histidines, in contrast to peptides with a higher number of histidines (e.g. His6 or HHHMVH), is explained based on the differences in the pH dependence of their affinity for copper. As discussed, the selected peptides with two histidines will be superior affinity tails than peptides with a higher histidine content (e.g. His6). Moreover, a peptide with a single histidine but with a very high copper affinity, is also identified. Its high copper affinity is related to the presence of several hydrophobic residues in the neighborhood of histidine. Chromatography of human interleukin-1 beta (hIL-1 beta) and several other proteins containing a single surface-exposed histidine surrounded by several hydrophobic residues confirmed that such a sequence could also serve as a very effective metal binding domain for protein purification using immobilized copper(II) columns.
Patwardhan
A V
AV
Department of Chemical Engineering, University of Pittsburgh, PA 15219, USA.
Goud
G N
GN
Koepsel
R R
RR
Ataai
M M
MM
D000345:Affinity Labels; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods; D003090:Coliphages / Q000235:genetics; D003300:Copper / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids; D007375:Interleukin-1 / Q000235:genetics / Q000302:isolation & purification; D019151:Peptide Library; D010455:Peptides / Q000235:genetics*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000345:Affinity Labels; D002614:Chelating Agents; D007098:Imino Acids; D007375:Interleukin-1; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D003300:Copper; C008109:iminodiacetic acid