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A V Patwardhan

Publications and source records attributed to A V Patwardhan.

9 recordsLinked to original sources
PubMed1997

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.

Affinity Labels↗
PubMed1997

Site accessibility and the pH dependence of the saturation capacity of a highly cross-linked matrix. Immobilized metal affinity chromatography of bovine serum albumin on chelating Superose.

Immobilized metal ion affinity chromatography has shown promise for isolating desired proteins from a mixture based on their affinity for chelated metal ions. Using frontal analysis, the pH dependence of the saturation capacity of chelating Superose matrix for bovine serum albumin (BSA) is examined over a broad pH range. A significant increase in the capacity was observed near the elution pH of BSA (pH 4.5) from a Cu-imminodiacetic acid column. The results of several experiments indicated that this apparently abnormal variation may reflect the low degree of accessibility of a large portion of copper sites inside chelating Superose. In a broader sense, these results suggest that during frontal analysis, the assumption of column saturation based on a plateau in the exit concentration that is almost at the same level as the input concentration could be misleading for highly cross-linked matrices and relatively large sized proteins. That is, the relatively less accessible copper sites may become difficult to be reached due to high levels of protein adsorption in the more accessible regions and thus give the appearance of a plateau in the breakthrough curve prior to complete column saturation. This is likely to be the case at high pH where BSA demonstrates very high affinity for immobilized copper or at high input concentrations where the equilibrium coverage is expected to be high. The results demonstrate that the estimated saturation capacity could be significantly smaller than the actual capacity.

Binding Sites↗