Expression, Purification And Properties Of Zinc Finger Protein ZNF191 (243-368)

There are about 3 billions base pairs in human genome, only 5% genome sequence can encode protein. But about 1% encoded human genome encode zinc finger proteins. And Cys₂His₂ (C₂H₂) zinc finger protein is the most typical class of zinc finger proteins. What is the biological function of C₂H₂ zinc finger protein super-family? A lot of researches show that many C₂H₂ zinc finger proteins function as important regulators in organisms by binding with DNA, RNA or proteins. But zinc finger proteins, especially crystals of zinc finger proteins are difficult to obtain, many basic questions of structure and biological function of this protein family still need to be studied furthermore.ZNF191 gene was obtained from a cDNA library of human liver, which was located at 18q12.1 of human chromosome, and this chromosomal region is related to many hereditary and tumor diseases, so ZNF191 gene possibly is also a correlative gene for some hereditary and tumor diseases. ZNF191 (243-368) is the zinc finger region protein of ZNF191 protein, containing four continuous C₂H₂ zinc finger domains, which probably can bind with specific DNA sequence and has specific gene regulation function. We attempt to identify the DNA sequence which can be specifically bounded by ZNF 191(243-368), and to understand the property and structure of each zinc finger of ZNF191 (243-368) as well as the role played by each zinc finger in whole ZNF191(243-368), therefore that will provide useful information for revealing the biological function of ZNF191.For studying the property, structure and function of a protein, a system which can stably and high-efficiently express the target protein with simple purification procedure is crucial. In experimental process, we found that the previous expression and purification of ZNF191(243-368) have many difficulties, especially the zinc finger-deletion mutants are difficult to be obtained. So we have tried four expression systems, i. e. pTSA-18/BL21(DE3), glutathione S-transferase (GST), His-tag and ketosteroid isomerase (KSI) expression systems. And we have characterized these proteins obtained from different expression systems.We constructed several recombinant plasmids containing genes of ZNF191(243-368) and its three zinc finger-deletion mutants (dF1, dF4, dF1F4) in pTSA-18/BL21(DE3) expression system. Though we can obtain ZNF191(243-368)from this system, its zinc finger-deletion mutants can not be obtained. And the yield of ZNF191(243-368) in 4 liters of medium is about 1 mg. It is insufficient for continuous researches, especially for the structure studies. So we try other expression system to obtain milligram quantities of ZNF191(243-368) and its zinc finger-deletion mutants.In glutathione S-transferase expression system the synergic expression of GST promotes the expression of ZNF191 (243-368), which makes the expression level of GST-ZNF191 (243-368) high. Based on the affinity of GST to Glutathione Sepharose 4B, we can obtain GST-ZNF 191 (243-368) simply by one step purification. We mixed the sepharose resin bounded GST-ZNF 191 (243-368) with a synthetic DNA oligonucleotide pool, and obtained the DNA oligonucleotide sequence which can tightly bind with ZNF191 (243-368) after repeated washing and amplification. By means of this experiment we obtained 69 independent clones among which 38 (55%) contained one AGGG base segment, 12 (17%) contained AGGA, AGGC and AGGT segment. The consensus binding sequence was suggested to be GGAGGG from the frequencies of the base at each position upon fixing of the "AGGG" core. Using this consensus binding sequence to search Eukaryotic Promoter Database we found many promoters of human genes contained this binding site which is in consistence with the fact that ZNF191 is widely expressed in human tissues. This result indicates that ZNF191 possibly has gene regulation function. Using fluorescence spectroscopy the binding ability of GST-ZNF 191 (243-368) to the obtained double-strand DNA sequence was also estimated by competition with EB. The result also showed that there was strong interaction between them. As reported GST can promote the crystallization of fusion protein, we hope to obtain the structure information of ZNF191(243-368) from the obtained GST fusion proteins.His-tag fusion expression system was developed in recent years. For the teminal of exogenous protein tagged a continuous histidine residues segment the target protein can be easily and simply purified by Ni²⁺-NTA resin. Some researches demonstrated that small His-tag does not affect the structure and function of the exogenous protein. So we constructed the genes of ZNF191 (243-368), its site-directed mutants and zinc finger-deletion mutants separately. At last, we obtained these proteins which are (His)₆-ZNF191 (243-368), (His)₆-F3/H4 (two cysteine residues of zinc finger 3 of ZNF191 (243-368) were mutated to His), (His)₆-F3/SS (two hydrophobic amino acids of zinc finger 3 were mutated to Ser)and ZNF191(243-368)-(His)₈. Unfortunately, we still can not obtain the zinc finger-deletion mutants for low expression level and instability of the zinc finger-deletion mutants in this system.The CD spectra of these proteins were visibly changed when zinc ion were removed, especially for the (His)₆-F3/SS protein. So zinc ion and hydrophobic amino acids residues are essential for sustaining the structure of zinc finger domain. When zinc ion was added zinc finger proteins can restore their original structures except the ZNF191(243-368)-(His)₈ protein. It is possible that His-tag at the C-terminal interferes this protein proper folding in vitro. We also found that (His)₆-F3/H4 and ZNF191(243-368)-(His)₈ possess the ability of hydrolytic cleavage DNA. This result indicates that four histidine residues coordinated with zinc ion can form a site which is similar with the active site of hydrolase. It is one of the possible reasons, explaining why in nature proteins do not contain H4-type zinc finger domains. The results indicated that use of His-tag expression system needs to be cautiously considered for zinc finger proteins which mainly depend on the coordination of Cys and His to zinc.We have identified the DNA-binding sequence of ZNF191 (243-368) and obtained several mutants, but still could not obtain the zinc finger-deletion mutants. In order to obtain zinc finger-deletion proteins and understand the difference among four zinc finger domains of ZNF191(243-368) we tried ketosteroid isomerase (KSI) expression system againWe constructed two genes of zinc finger 3 (ZF3) and 4 (ZF4) of ZNF191 together with ketosteroid isomerase gene in pET-31b vector separately, and overexpressed the fusion proteins in the form of inclusion bodies in E. coli. BL21(DE3)pLysS strain because of the insolubility and synergic expression of KSI. The inclusion body was purified by washing several times and its content of the ZF3 (or ZF4) is more than 80%, which can directly be cleaved by cyanogen bromide to release single zinc finger peptides. Based on the differences of molecular weight of KSI and single zinc finger, we can obtain single zinc finger peptide whose purity is more than 95% and yield is more than 20 mg/L by Amicon YM films filtration. The circular dichroism spectra of two single zinc finger peptides titrated by Zn (II) demonstrated that they have different secondary structure, and zinc ion is necessary for peptide folding. So this expression system provides us a suitable method for expression and purification of small peptides, and provides abundant peptide forproperty studies.From the studies of four expression systems we understand the expression and purification of protein need to be groped by a lot of experiments. While we tried these expression systems we identified the DNA-binding sequence of ZNF191 (243-368) using the affinity of GST in fusion protein to resin. We knew that zinc ion and hydrophobic amino acid residues were necessary for sustaining the structure of zinc finger domains and obtained two proteins having the ability of hydrolytic cleavage DNA from His-tag fusion proteins. We also successfully obtained zinc finger 3 and zinc finger 4 of ZNF191(243-368) using KSI expression system, and found these two zinc fingers had different structural property. These results deepen our understanding about the relationship among property, structure and function of ZNF191(243-368).