Expression and purification of a novel ZNF191 zinc finger protein

Liu, Yuqi; Liu, Yu; Yu, Wenhao; Shi, Shaolin; Sun, Bingyun; Wu, Guojun; Huang, Zhong‐Xian

doi:10.1007/bf02874239

Cited by 4 publications

(8 citation statements)

References 28 publications

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“…5 Fusion proteins could be partly purified after a single affinity chromatography step. And GST fusion proteins are more specific to the glutathione-sepharose 4B resin than the polyhistidine fusion proteins to Ni-NTA resin.…”

Section: Purification Of Proteinsmentioning

confidence: 99%

“…Inclusion body expression system: employing PCR (polymerase chain reaction) strategy, we used pTSA-ZNF191 plasmid as a template, 5 an up-primer P1: 5'-GGAATTCCATATGAGAAA-TCCCTCTCGAAAG AAACA-3' (introduced a 5' Nde I site) and a downprimer P2: 5'-GCCCAAGCTTAAACTTCCCAACA-TTCAG-3' (introducing a 3' Hind III site) to obtain the zinc finger gene. Then the inserted zinc finger gene and pTSA-18 vector were digested with Nde I and Hind III restriction enzymes, separated by electrophoresis in agarose.…”

Section: Plasmid Constructionsmentioning

confidence: 99%

“…for the 6×His expression system (the purification of the inclusion body was as mentioned before 5 ). And cells were lysed by lysozyme for about 1 h at 4 ℃, then treated with 5 U/mL DNase and stirred for 30 min to degenerate nucleic acids at 4 ℃.…”

Section: Purification Of Proteinsmentioning

confidence: 99%

“…3,4 ZNF191 (243-368) is the zinc finger region of the ZNF191 protein, including four continuous Cys 2 -His 2 zinc finger motifs, which is a putative DNA binding domain. 5 But its actual function and DNA-binding site remain unknown.…”

Section: Introductionmentioning

confidence: 99%

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

Zhao¹,

Teng²,

Ding³

et al. 2007

Chin. J. Chem.

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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.

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Section: Purification Of Proteinsmentioning

confidence: 99%

Section: Plasmid Constructionsmentioning

confidence: 99%

Section: Purification Of Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Zhao¹,

Teng²,

Ding³

et al. 2007

Chin. J. Chem.

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“…The ZNF191 gene is identified from a cDNA library derived from human liver. This gene is located on human chromosome 18q12.1 and is related to a few heredity and tumor diseases [ 13 , 14 ]. ZNF191 encodes a 368-amino acid protein which includes a putative DNA-binding domain of four Cys 2 His 2 zinc finger motifs at the C-terminal region.…”

Section: Introductionmentioning

confidence: 99%

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

Zhao

Huang

2015

Bioinorganic Chemistry and Applications

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ZNF191(243-368) is the C-terminal region of ZNF191 which contains a putative DNA-binding domain of four Cys2His2 zinc finger motifs. In this study, an expression vector of a fusion protein of ZNF191(243-368) with glutathione-S-transferase (GST) was constructed and transformed into Escherichia coli BL21. The fusion protein GST-ZNF191(243-368) was expressed using this vector to investigate the protein-DNA binding reaction through an affinity selection strategy on the basis of the binding quality of the zinc finger domain. Results showed that ZNF191(243-368) can selectively bind with sequences and react with genes which contain an AGGG core. However, the recognition mechanism of Cys2His2 zinc finger proteins to DNA warrants further investigation.

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Wnt8B, transcriptionally regulated by ZNF191, promotes cell proliferation of hepatocellular carcinoma via Wnt signaling

Liu

Cheng

et al. 2020

Cancer Science

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Dysregulation of wingless‐type (Wnt) signaling is implicated in hepatocellular carcinoma (HCC). Wnt family member 8B (Wnt8B), one of the canonical Wnt ligands, is implicated in oncogenesis. However, the role of Wnt8B in human HCCs and its transcriptional regulation mechanism are presently unknown . Here, we report that Wnt8B expression was frequently increased in HCCs and was significantly associated with poorer patient prognosis. Wnt8B knockdown suppresses HCC cell growth both in vitro and in vivo via inhibiting the canonical Wnt signaling. Zinc finger transcription factor 191 (ZNF191) can positively regulate Wnt8B mRNA and protein expression, and promoter luciferase assay indicated that ZNF191 can increase the transcription activity of the 2‐Kbps WNT8B promoter. Chromatin immunoprecipitation‐qPCR and electrophoretic mobility shift assay showed that ZNF191 protein directly binds to the WNT8B promoter, and the binding sites are at nt‐1491(ATTAATT) and nt‐1178(ATTCATT). Moreover, Wnt8B contributes to the effect of ZNF191 on cell proliferation, and Wnt8B expression correlates positively with ZNF191 in human HCCs. Our findings suggested that Wnt8B, directly transcriptionally regulated by ZNF191, plays a pivotal role in HCC proliferation via the canonical Wnt pathway and may serve as a new prognostic biomarker and a potential therapeutic target for HCC patients.

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Expression and purification of a novel ZNF191 zinc finger protein

Cited by 4 publications

References 28 publications

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

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

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

Wnt8B, transcriptionally regulated by ZNF191, promotes cell proliferation of hepatocellular carcinoma via Wnt signaling

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