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

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Dongxin Zhao ◽  
Zhongxian Huang
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression System ◽  
Zinc Finger Proteins ◽  
Hereditary Diseases ◽  
Expression Systems ◽  
His Tag ◽  
Finger Protein ◽  
Inclusion Body Formation ◽  
Structure And Functions

Zinc finger proteins are associated with hereditary diseases and cancers. To obtain an adequate amount of zinc finger proteins for studying their properties, structure, and functions, many protein expression systems are used. ZNF191(243-368) is a zinc finger protein and can be fused with His-tag to generate fusion proteins such as His6-ZNF191(243-368) and ZNF191(243-368)-His8. The purification of His-tag protein using Ni-NTA resin can overcome the difficulty of ZNF191(243-368) separation caused by inclusion body formation. The influences of His-tag on ZNF191(243-368) properties and structure were investigated using spectrographic techniques and hydrolase experiment. Our findings suggest that insertion of a His-tag at the N-terminal or C-terminal end of ZNF191(243-368) has different effects on the protein. Therefore, an expression system should be considered based on the properties and structure of the protein. Furthermore, the hydrolase activity of ZNF191(243-368)-His8has provided new insights into the design of biological functional molecules.

Single zinc-finger extension: enhancing transcriptional activity and specificity of three-zinc-finger proteins

2005 ◽  
Vol 386 (2) ◽  
pp. 95-99 ◽  
Author(s):  
Alexander E.F. Smith ◽  
Farzin Farzaneh ◽  
Kevin G. Ford
Keyword(s):  
Transcription Factors ◽  
Fusion Protein ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Finger Protein ◽  
Finger Extension ◽  
Vp16 Fusion

AbstractIn order to demonstrate that an existing zinc-finger protein can be simply modified to enhance DNA binding and sequence discrimination in both episomal and chromatin contexts using existing zinc-finger DNA recognition code data, and without recourse to phage display and selection strategies, we have examined the consequences of a single zinc-finger extension to a synthetic three-zinc-finger VP16 fusion protein, on transcriptional activation from model target promoters harbouring the zinc-finger binding sequences. We report a nearly 10-fold enhanced transcriptional activation by the four-zinc-finger VP16 fusion protein relative to the progenitor three-finger VP16 protein in transient assays and a greater than five-fold enhancement in stable reporter-gene expression assays. A marked decrease in transcriptional activation was evident for the four-zinc-finger derivative from mutated regulatory regions compared to the progenitor protein, as a result of recognition site-size extension. This discriminatory effect was shown to be protein concentration-dependent. These observations suggest that four-zinc-finger proteins are stable functional motifs that can be a significant improvement over the progenitor three-zinc-finger protein, both in terms of specificity and the ability to target transcriptional function to promoters, and that single zinc-finger extension can therefore have a significant impact on DNA zinc-finger protein interactions. This is a simple route for modifying or enhancing the binding properties of existing synthetic zinc-finger-based transcription factors and may be particularly suited for the modification of endogenous zinc-finger transcription factors for promoter biasing applications.

scholarly journals A Krüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

2002 ◽  
Vol 22 (12) ◽  
pp. 4256-4267 ◽  
Author(s):  
Kazuhiro Tanaka ◽  
Noriyuki Tsumaki ◽  
Christine A. Kozak ◽  
Yoshihiro Matsumoto ◽  
Fumihiko Nakatani ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Promoter Activity ◽  
Expression Vector ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Limb Bud ◽  
Collagen Gene ◽  
Specific Expression ◽  
Finger Protein ◽  
Krab Domain

ABSTRACT Type XI collagen is composed of three chains, α1(XI), α2(XI), and α3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the −530-bp promoter segment of the α2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

The Regulation of Zinc Finger Proteins by Mirnas Enriched in ALL-Microvesicles

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1448-1448
Author(s):  
Huiyu Li ◽  
Xiaomei Chen ◽  
Wei Xiong ◽  
Fang Liu ◽  
Shiang Huang
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression Profiles ◽  
Zinc Fingers ◽  
Zinc Finger Proteins ◽  
Bioactive Molecules ◽  
Chromosomal Protein ◽  
Normal Peripheral Blood ◽  
Finger Protein

Abstract Abstract 1448 Microvesicles (MVs) are submicrometric membrane fragments and they can “hijack” membrane components and engulf cytoplasmic contents from their cellular origin. MVs are enriched in various bioactive molecules of their parental cells, such as proteins, DNA, mRNA and miRNAs. Microvesicles (MVs) released by leukemia cells constitute an important part of the leukemia microenvironment. As a cell-to-cell communication tool, MVs transfer microRNA (miRNA) between cells. MVs miRNAs may also provide an insight in the role of miRNAs playing in the underlying of pathophysiologic processes of various leukemia. We determined the miRNA expression profiles of ALL-derived MVs using Agilent miRNA microarray analysis. The five miRNAs obtained by microarray profiling were validated using real-time PCR. The putative target genes were predicted by bioformation software. We identified 182 and 166 dysregulated miRNAs in MVs derived from Nalm 6 cells and from Jurkat cells, respectively. Both up regulated (123/182 in Nalm 6-MVs and 114/166 in Jurkat- MVs) and down regulated (59/182 in Nalm 6-MVs and 52/166 in Jurkat- MVs) expressions were observed compared with MVs from normal peripheral blood the MVs normal control. When we analyzed those miRNA with bioinformatic tools (TargetScan), we found an interesting phenomenon that presence of 111 zinc fingers genes were regulated by 52 miRNAs, indicating that the ALL-microvesicles were enriched with miRNAs regulating zinc finger proteins. They encompassed zinc fingers and homeoboxes 2, zinc finger, ZZ-type containing 3, zinc finger, SWIM-type containing 1, zinc finger, RAN-binding domain containing 3, zinc finger, NFX1-type containing 1, zinc finger, MYM-type 4, zinc finger, FYVE domain containing 1 and their 5 subtypes; zinc finger, DHHC-type containing16, and other subtypes; zinc finger, CCHC domain containing 14 and 7A, zinc finger, BED-type containing 4; zinc finger protein, X-linked; zinc finger protein, multitype 2; zinc finger protein 81, and their 55 subtypes; zinc finger and SCAN domain containing 18, zinc finger and BTB domain containing 9. ALL-microvesicles were enriched with expression changes of distinct sets of miRNAs regulating zinc finger proteins. This provides clues that genes commonly function together. It is worth noting that 52 miRNA regulating above zinc finger protein genes were up-expressed, suggeting that miRNA regulating zinc fingers were active in ALL-MVs. Zinc finger proteins are important transcriptions in eukaryotes and play roles in regulating gene. Some members of the Zinc finger family have close relationaship with tumour. Zinc finger X-chromosomal protein (Zfx) is a protein that in humans is encoded by the ZFX gene. The level of Zfx expression correlates with aggressiveness and severity in many cancer types, including prostate cancer, breast cancer, gastric tumoural tissues, and leukemia. [1,2]. Zinc finger and homeoboxes 2 (ZHX2) was target gene of miRNA-1260. The role of miRNA are negatively regulated host gene expressions. ZHX2 inhibits HCC cell proliferation by preventing expression of Cyclins A and E, and reduces growth of xenograft tumors. Loss of nuclear ZHX2 might be an early step in the development of HCC[3]. In our study, the miRNA-1260 were 9 fold higher in ALL MVs. In leukeima microenvironment, ALL-MVs may transfer aberantly expressed miRNAs to their target cell lead to abnormally regulated the zinc finger proteins that may play roles in ALL. In this study, we demonstrated that ALL-microvesicles were enriched with expression changes of distinct sets of miRNAs regulating zinc finger proteins. Futhermore, Zinc fingers were active in ALL-MVs and commonly function together. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of Zinc Finger Protein Zat12 from Arabidopsis thaliana in Escherichia coli

2020 ◽  
Vol 3 (1) ◽  
pp. 504-511
Author(s):  
Le Thi Tuyet Cham ◽  
Vu Ngoc Thang ◽  
Tran Anh Tuan ◽  
Vu Thi Thuy Hang
Keyword(s):  
Arabidopsis Thaliana ◽  
Zinc Finger ◽  
Stress Responses ◽  
Zinc Finger Protein ◽  
Expression System ◽  
Direct Interaction ◽  
Early Response ◽  
In Planta ◽  
Abiotic Stress Response ◽  
Finger Protein

The C2H2 zinc finger protein ZAT12 has been classified as a plant core abiotic stress response gene in the early  response to multiple stresses. ZAT12 links the iron deficiency and oxidative stress responses through the direct interaction with/and negative regulation of a central regulator - FIT. For further research on the regulation of the ZAT12 protein in planta, a huge quantity of ZAT12 proteins is required to inject into mice for the generation of ZAT12 antiserum. In this study, the gene encoding the ZAT12 protein from Arabidopsis thaliana was cloned into the expression vector - pETBlue-2 and then overexpressed in E. coli T7. A high expression level was indicated by SDS-PAGE. Immunoblot demonstrated successful expression using a bacterial expression system.

scholarly journals The Barley Stripe Mosaic Virus expression system reveals the wheat C2H2 zinc finger protein TaZFP1B as a key regulator of drought tolerance

2019 ◽  
Author(s):  
Mario Houde ◽  
Arnaud Cheuk ◽  
François Ouellet
Keyword(s):  
Drought Stress ◽  
Drought Tolerance ◽  
Mosaic Virus ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Barley Stripe Mosaic Virus ◽  
Finger Protein

Abstract Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speeds up functional gene characterization.Results We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant’s life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected.Conclusions This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat.

scholarly journals Correlation between functional and binding activities of designer zinc-finger proteins

2007 ◽  
Vol 403 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Jong Seok Kang
Keyword(s):  
Binding Affinity ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Sequence Specificity ◽  
Binding Assays ◽  
Finger Protein ◽  
Vitro Binding

Rapid progress in the ability to develop and utilize zinc-finger proteins with customized sequence specificity have led to their increasing use as tools for modulation of target gene transcription in the post-genomic era. In the present paper, a series of in vitro binding assays and in vivo reporter analyses were used to demonstrate that a zinc-finger protein can effectively specify a base at each position of the target site in vivo and that functional activity of the zinc-finger protein as either a transcriptional repressor or activator is positively correlated with its binding affinity. In addition, this correlation can be extended to artificial engineered zinc-finger proteins. These data suggest that the binding affinity of designer zinc-finger proteins with novel specificity might be a determinant for their ability to regulate transcription of a gene of interest.

scholarly journals The Barley Stripe Mosaic Virus expression system reveals the wheat C2H2 zinc finger protein TaZFP1B as a key regulator of drought tolerance

2020 ◽  
Author(s):  
Arnaud Cheuk ◽  
François Ouellet ◽  
Mario Houde
Keyword(s):  
Drought Stress ◽  
Drought Tolerance ◽  
Mosaic Virus ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Barley Stripe Mosaic Virus ◽  
Finger Protein

Abstract Background Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speeds up functional gene characterization. Results We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant’s life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected. Conclusions This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat.

scholarly journals Specific Recognition of ZNF217 and Other Zinc Finger Proteins at a Surface Groove of C-Terminal Binding Proteins

2006 ◽  
Vol 26 (21) ◽  
pp. 8159-8172 ◽  
Author(s):  
Kate G. R. Quinlan ◽  
Marco Nardini ◽  
Alexis Verger ◽  
Pierangelo Francescato ◽  
Paul Yaswen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Transcription Factors ◽  
Zinc Finger ◽  
Target Gene ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Repressor Protein ◽  
Finger Protein ◽  
Surface Groove ◽  
Binding Cleft

ABSTRACT Numerous transcription factors recruit C-terminal binding protein (CtBP) corepressors. We show that the large zinc finger protein ZNF217 contacts CtBP. ZNF217 is encoded by an oncogene frequently amplified in tumors. ZNF217 contains a typical Pro-X-Asp-Leu-Ser (PXDLS) motif that binds in CtBP's PXDLS-binding cleft. However, ZNF217 also contains a second motif, Arg-Arg-Thr (RRT), that binds a separate surface on CtBP. The crystal structure of CtBP bound to an RRTGAPPAL peptide shows that it contacts a surface crevice distinct from the PXDLS binding cleft. Interestingly, both PXDLS and RRT motifs are also found in other zinc finger proteins, such as RIZ. Finally, we show that ZNF217 represses several promoters, including one from a known CtBP target gene, and mutations preventing ZNF217's contact with CtBP reduce repression. These results identify a new CtBP interaction motif and establish ZNF217 as a transcriptional repressor protein that functions, at least in part, by associating with CtBP.

scholarly journals Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins

The Analyst ◽  
2015 ◽  
Vol 140 (12) ◽  
pp. 3947-3952 ◽  
Author(s):  
Soodong Noh ◽  
Dat Thinh Ha ◽  
Haesik Yang ◽  
Moon-Soo Kim
Keyword(s):  
Alkaline Phosphatase ◽  
Electrochemical Detection ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Direct Detection ◽  
Electrochemical Measurement ◽  
Zinc Finger Proteins ◽  
Double Stranded Dna ◽  
Ito Electrode ◽  
Finger Protein

We report sensitive and direct detection of specific bacterial dsDNA sequences using (i) the zinc finger protein labelled with alkaline phosphatase and (ii) electrochemical measurement at the ITO electrode.

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