Expression and Purification of ZNF191(243–368) in Three Expression Systems

Abstract

ZNF191(243-368), a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain adequate amount of ZNF191(243-368) for the study of its property, structure and function, three different expression systems of inclusion-body, glutathione S-transferase (GST), and hexahistidine (6×His) were used and compared. Among these systems, the expression level of ZNF191(243-368) was increased in inclusion body system under a higher isopropylthio-β-D-galactoside (IPTG) concentration, but the non-target proteins were also increased more, which made its purification more difficult and the yield lower. The expression of His-tag fusion protein was almost not affected by IPTG concentration, temperature and inducing time. At a high IPTG concentration the highest expression yield for GST fusion protein was obtained. And the fusion proteins can be partially purified by a single affinity chromatography step. The fusion protein systems show advantages for expression of these proteins.

Recognition Code of ZNF191(243-368) and Its Interaction with DNA

“…The zinc finger protein more strongly binds to DNA1 than to DNA2. In addition, the binding result of DNA2 is close to previous studies, in which two mutant proteins of ZNF191(243-368) were purified from the pTSA/BL21(DE3) system [ 15 ]. Thus, the fusion protein can be used to study protein-DNA interactions by the fluorescence method of the three-component system with the highly sensitive and selective EB fluorescence probe.…”

“…The ligation product was transformed into BL21 host strains. The bacterial clone was sequenced, and the recombinant vector was named pGEX-ZF [ 15 ].…”

Effect of His-Tag on Expression, Purification, and Structure of Zinc Finger Protein, ZNF191(243-368)

“…The pQE-ZF plasmid was constructed as previously described for expressing His 6 -ZNF191(243-368) [ 9 ]. The primers used to construct the ZNF191(243-368)-His 8 expression system were as follows: up-primer: 5′-GGAATTC CATATG AGAAATCCCTCTCGAAAGAAACA-3′; down-primer: 5′-CCG CTCGAG AACTTCCACAACATTCAGAAG-3′.…”

“…High expression of proteins was induced under optimum conditions. The expression and purification of ZNF191(243-368)-His 8 were similar to those of His 6 -ZNF191(243-368) [ 9 ]. The cells harvested from 500 mL of LB medium grown and induced under the optimum conditions were suspended in 10 volumes of cell lysis buffer (50 mmol·L −1 NaH 2 PO 4 , pH 8.0, 300 mmol·L −1 NaCl, and 5 mmol·L −1 imidazole).…”