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Rapid and Efficient Recovery of Histidine‐tagged Proteins Directly from Bacterial Cultures
Author(s) -
Porter Jeffrey John,
Mehigh Richard,
Boyle Joanne Akiko,
Chen Dian Er,
Kappel William,
Scott Graham Bruce Ian
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a530-d
Subject(s) - lysis , protein purification , chromatography , downstream processing , affinity chromatography , cell disruption , recombinant dna , centrifugation , chemistry , tandem affinity purification , target protein , histidine , fractionation , biochemistry , enzyme , gene
The purification of recombinant proteins is often a time‐consuming and tedious process. Traditional protein purification methods from E.coli require an initial harvesting of cells, which are then subjected to mechanical, detergent, or enzymatic lysis to solubilize cellular protein(s). The crude cell lysates must then be clarified by centrifugation before applying to an affinity resin for purification. In order to overcome this bottleneck in proteomics research, Sigma‐Aldrich has developed the HIS‐SelectTM iLAPTM (Integrated Lysis and Affinity Purification) 5 ml Column. The HIS‐Select iLAP 5 ml Column is a single‐use, disposable column designed for one‐step purification of histidine‐tagged proteins directly from a 5 ml bacterial culture. By eliminating the cell harvest and sample clarification steps, the iLAP technology streamlines the entire purification process, allowing for rapid recovery of the target protein(s). When compared to traditional methods, this improved procedure yields similar results, and requires far less time and effort. Additionally, the resulting purified protein is suitable for a variety of downstream applications. In the present work, we have utilized the HIS‐Select iLAP columns for the purification of a variety of Metal Affinity Tag (MAT) proteins, and demonstrated the utility of the HIS‐Select iLAP 5 ml Column for screening the expression level of a variety of bacterial clones expressing MAT‐tagged proteins.