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Quantitative evaluation of His‐tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells
Author(s) -
Cao Heping,
Lin Rui
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.121
Subject(s) - immunoprecipitation , tristetraprolin , transfection , recombinant dna , mutant , microbiology and biotechnology , hek 293 cells , affinity chromatography , phosphorylation , chemistry , myc tag , biochemistry , blot , protein purification , biology , fusion protein , gene , messenger rna , untranslated region , enzyme
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 [ 32 P]‐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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009