Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

Authors: Cao, Heping; LIN, RUI - NIH-NIEHS, NC

Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/25/2008
Publication Date: 3/1/2009
Citation: Cao, H., Lin, R. 2009. Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells. Biotechnology Progress. 25:461-467.

Interpretive Summary: Recombinant protein production is an important biotechnological process for generating sufficient amounts of pure proteins otherwise difficult to obtain from natural sources. Purified recombinant proteins are widely used for producing research reagents by academic, governmental, and industrial laboratories. One of the most commonly used procedures is the histidine (His)-tag affinity purification system. This system generates fusion proteins consisting of proteins of interest and multiple histidine residues for facilitating protein purification by affinity chromatography. For example, the His-tag procedure was used to express tristetraprolin/zinc finger protein 36 (TTP/ZFP36) in E. coli and human cells. The popularity of this method is due, in part, to its many advantageous properties, such as the high affinity of the His-tag in recombinant protein with purification systems. In addition, the small size of His-tag generally does not interfere with biochemical activities of the tagged proteins. However, the yield and purity of various proteins purified by this procedure are quite different and require quantitative evaluation of efficiency. Our results demonstrated that the His-tag purification procedure is more effective than other commonly used procedures. These results will be important to scientists working to improve purification procedures for research reagents used to understand the molecular mechanisms related to nutrition.

Technical Abstract: 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 the current study was to evaluate the His-tag procedure quantitatively and to compare it with immunoprecipitation using radiolabeled tristetraprolin (TTP), a zinc finger protein with anti-inflammatory properties. 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 [32P]-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.