4.3 Article

Quantitative Evaluation of His-Tag Purification and Immunoprecipitation of Tristetraprolin and its Mutant Proteins from Transfected Human Cells

Journal

BIOTECHNOLOGY PROGRESS
Volume 25, Issue 2, Pages 461-467

Publisher

WILEY
DOI: 10.1002/btpr.121

Keywords

His-tag purification; immunoprecipitation; in vivo radiolabeling; phosphorylation site; site-directed mutagenesis; tristetraprolin; zinc finger protein

Funding

  1. NIH
  2. National Institute of Environmental Health Sciences
  3. USDA-ARS Human Nutrition Research Program

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Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of this study was to evaluate His-tag procedure quantitatively and to compare it with immunoprecipitation using radiolabeled tristetraprolin (TTP), a zinc finger protein with anti-inflammatory property. Human embryonic kidney 293 cells were transfected with wild-type and nine mutant plasmids with single or multiple phosphorylation site mutation(s) in His-TTP. These proteins were expressed and mainly localized in the cytosol of transfected cells by immunocytochemistry and confocal microscopy. His-TTP proteins were purified by Ni-NTA beads with imidazole elution, or precipitated by TTP antibodies from transfected cells after being labeled with [P-32]-orthophosphate. The results showed that (1) His-tag purification was more effective than immunoprecipitation for TTP purification; (2) mutations in TTP increased the yield of His-TTP by both purification procedures; and (3) mutations in TTP increased the binding affinity of mutant proteins for Ni-NTA beads. These findings suggest that bioengineering phosphorylation sites in proteins can increase the production of recombinant proteins. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 461-467, 2009

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