Specific N-methylations of HPV-16 E7 peptides alter binding to the retinoblastoma suppressor protein.

Complex formation between the human papilloma virus type 16 E7 protein (HPV-16 E7) and the retinoblastoma growth suppressor protein (RB) is believed to contribute to the process of cellular transformation that leads to cervical carcinoma. Genetic analysis of the HPV-16 E7 protein has shown that the segment of E7 homologous to the conserved region 2 of adenovirus 5 E1A protein is involved in both RB binding and E7-mediated cell transformation. We have previously shown that a peptide colinear with HPV-16 E7 residues 21-29 was able to block immobilized species of E7 from binding to RB protein. The current study reports the effects of different chemical modifications of this peptide. One type of modification, methylation of the alpha-amino nitrogens contributed by Leu22, Tyr25, and Leu28, resulted in a 45-fold increase in E7/RB binding antagonist activity. This increased antagonist activity is sequence-specific since methylation of the amino groups contributed by Tyr23, Cys24, or Glu26 resulted in a profound loss of binding antagonist activity. Using a newly developed binding assay we determined that the apparent dissociation constant for recombinant HPV-16 E7 protein binding to recombinant human RB protein is 1.3 nM. The peptide Ac[N-MeLeu22,N-Me-Tyr25,N-MeLeu28]-(21-29)-E7 amide was determined to be a competitive inhibitor of HPV-16 E7 binding to RB with a Ki value of 32 nM.     

The human DEK proto-oncogene is a senescence inhibitor and an upregulated target of high-risk human papillomavirus E7

The human DEK proto-oncogene is a nucleic acid binding protein with suspected roles in human carcinogenesis, autoimmune disease, and viral infection. Intracellular DEK functions, however, are poorly understood. In papillomavirus-positive cervical cancer cells, downregulation of viral E6/E7 oncogene expression results in cellular senescence. We report here the specific repression of DEK message and protein levels in senescing human papillomavirus type 16- (HPV16-) and HPV18-positive cancer cell lines as well as in primary cells undergoing replicative senescence. Cervical cancer cell senescence was partially overcome by DEK overexpression, and DEK overexpression was sufficient for extending the life span of primary keratinocytes, supporting critical roles for this molecule as a senescence regulator. In order to determine whether DEK is a bona fide HPV oncogene target in primary cells, DEK expression was monitored in human keratinocytes transduced with HPV E6 and/or E7. The results identify high-risk HPV E7 as a positive DEK regulator, an activity that is not shared by low-risk HPV E7 protein. Experiments in mouse embryo fibroblasts recapitulated the observed E7-mediated DEK induction and demonstrated that both basal and E7-induced regulation of DEK expression are controlled by the retinoblastoma protein family. Taken together, our results suggest that DEK upregulation may be a common event in human carcinogenesis and may reflect its senescence inhibitory function.     

Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli

Glucarpidase (former name: carboxypeptidase G2, or CPG2) is a bacterial enzyme that is widely used in detoxification of the cytotoxic drug, methotrexate, and in Antibody Directed Enzyme Prodrug Therapy for cancer treatment. The glucarpidase gene of Pseudomonas sp. strain RS-16 was previously cloned in E coli, but expresses at a level that is approximately 100-fold lower than in the native strain. In this study, a synthetic gene coding for glucarpidase was codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work indicated that the enzyme was expressed to ~60% of the total host protein and that purification of the recombinant His-tagged protein could be achieved in a single step by Ni²⁺ charged column chromatography. The synthetic recombinant glucarpidase expressed within this system was biologically active and zinc dependant. Our study showed that Mg²⁺ as well as Mn²⁺ ions inhibit the activity of the recombinant enzyme.     

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.     

High-level production of recombinant chicken interferon-gamma by Brevibacillus choshinensis

Cytokines, such as interferon-gamma have been shown to have adjuvant and growth promoting activity in poultry and livestock and have the potential to be used as alternatives to antibiotics. We have developed an efficient system for commercial-scale synthesis of recombinant chicken interferon-gamma (ChIFN-gamma) using Brevibacillus choshinensis as the host for protein production. The ChIFN-gamma expression vector, pNCIFN, was constructed using the novel Escherichia coli-B. choshinensis shuttle vector, pNCMO2. ChIFN-gamma expression was optimized by investigating different culture conditions and different host B. choshinensis mutants. The highest level of production was observed using the B. choshinensis HPD31-MB2 strain grown at 30 degrees C, where ChIFN-gamma was produced at approximately 300-500 mg/L. ChIFN-gamma was also produced as a His-tagged fusion protein by using the pNCHis-IFN expression vector, a derivative of pNCMO2. The protein was constitutively secreted into the culture supernatant and could be partially purified in a single step using a Ni-nitrilotriacetic acid column. This recombinant His-ChIFN-gamma was shown to have the same biological activity as native ChIFN-gamma.     

A fluorogenic substrate as quantitative in vivo reporter to determine protein expression and folding of tobacco etch virus protease in Escherichia coli

Quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including Escherichia coli. To determine the yield of soluble active protease from the tobacco etch virus (TEV), we developed a single-molecule assay based on the fluorogenic substrate ANA-QS-MCA. This substrate consists of a 10 amino acid peptide (ENLYFQSGTK) containing the proteolytic cleavage sequence of the TEV protease. The peptide works as a linker N-terminally tagged with a fluorescent donor group (7-Methoxycoumarin-4-yl)acetyl (MCA) and C-terminally tagged with the acceptor group 5-Amino-2-nitrobenzoic acid (ANA). Fluorescence can be observed after specific cleavage of the substrate at the Gln-Ser bond by active TEV protease. Purified His-tagged TEV protease was used for in vitro analysis. Through determination of proteolytic activity in living E. coli cells and through application of Confocal Laser-Scanning-Microscopy we demonstrate that the peptide is well suited to in vivo expression analysis. This provides an effective tool to monitor the accumulation of active recombinant TEV protease in crude extracts and intact cells.     

Expression and purification of a biologically active Phytophthora parasitica cellulose binding elicitor lectin in Pichia pastoris

The Phytophthora parasitica cellulose-binding elicitor lectin, (CBEL), is a cell wall-localized protein playing a key role in cell wall organization and adhesion of the mycelium to cellulosic substrates. CBEL is a potent elicitor of plant immune responses and this activity is linked to its ability to bind plant cell wall components. In order to scale up the production of active CBEL, we reported here the cloning and expression of a His-tagged version of CBEL in the yeast Pichia pastoris. Selection of a high-producing P. pastoris clone and optimization of the purification procedure allowed a yield of about 2mg of pure protein per liter of culture filtrate. The identity of the recombinant protein was confirmed by western-blot analysis, N-terminal protein sequencing, and by peptide mass fingerprinting. The cellulose-binding affinity and the lectin activity of the recombinant protein were identical to the native CBEL. Its elicitor activity, tested on Arabidopsis thaliana leaves, was similar to the native CBEL protein as it displays a similar biological activity on plant immune responses inducing defense gene expression and localized necroses of the infiltrated leaf tissues. The present work suggests that P. pastoris can be a suitable host for the production of compounds active on plants or for the development of new agricultural products able to stimulate plant immunity.     

The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.     

High-risk human papillomavirus E5 oncoprotein displays channel-forming activity sensitive to small-molecule inhibitors

High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and therefore is responsible for significant morbidity and mortality worldwide. Cellular transformation is mediated directly by the expression of viral oncogenes, the least characterized of which, E5, subverts cellular proliferation and immune recognition processes. Despite a growing catalogue of E5-specific host interactions, little is understood regarding the molecular basis of its function. Here we describe a novel function for HPV16 E5 as an oligomeric channel-forming protein, placing it within the virus-encoded "viroporin" family. The development of a novel recombinant E5 expression system showed that E5 formed oligomeric assemblies of a defined luminal diameter and stoichiometry in membranous environments and that such channels mediated fluorescent dye release from liposomes. Hexameric E5 channel stoichiometry was suggested by native PAGE studies. In lieu of high-resolution structural information, established de novo molecular modeling and design methods permitted the development of the first specific small-molecule E5 inhibitor, capable of both abrogating channel activity in vitro and reducing E5-mediated effects on cell signaling pathways. The identification of channel activity should enhance the future understanding of the physiological function of E5 and could represent an important target for antiviral intervention.     

Mouse methionine sulfoxide reductase B: effect of selenocysteine incorporation on its activity and expression of the seleno-containing enzyme in bacterial and mammalian cells

The mammalian methionine sulfoxide reductase B (MsrB) has been found to be a selenoprotein which can reduce R form of both free and protein-incorporated methionine sulfoxide to methionine. Together with MsrA, which reduces specifically the S form of methionine sulfoxide, the living cell can repair methionine-damaged proteins and salvage free methionine under oxidative stress conditions. Here, we report about the pivotal role of the selenocysteine residue in the protein putative active site by site-directed mutagenesis directed to the selenocysteine codon. Using the Escherichia coli SECIS (selenocysteine insertion sequence) element, needed for the recognition of the UGA codon as a selenocysteine codon in E. coli, we expressed the seleno-MsrB as a recombinant selenoprotein in E. coli. The recombinant seleno-MsrB has been shown to be much more active than the cysteine mutant, whereas the mutations to alanine and serine rendered the protein inactive. Although the yields of expression of the full-length N-terminus and C-terminus His-tagged seleno-MsrB were only 3% (of the total MsrB expressed), the C-terminus His-tagged protein enabled us to get a pure preparation of the seleno-MsrB. Using both recombinant selenoproteins, the N-terminus His-tagged and the C-terminus His-tagged proteins, we were able to determine the specific activities of the recombinant seleno-MsrB, which were found to be much higher than the cysteine mutant homologue. This finding confirmed our suggestion that the selenocysteine is essential for maintaining high reducing activity of MsrB. In addition, using radioactive selenium we were able to determine the in vivo presence of MsrB as a selenoprotein in mammalian cell cultures.     

A synthetic E7 gene of human papillomavirus type 16 that yields enhanced expression of the protein in mammalian cells and is useful for DNA immunization studies

A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20- to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G + C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system.     

Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta

We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.     

Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography

Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.     

大肠杆菌抗氟乙酸变株的选育及应用

In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E.coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon…     

Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli.

We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding alpha-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO). The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g(-1) of cell (and a fermentation yield of 3.82 U x L(-1) of fermentation broth). An N-terminal His-tagged protein (HisGO) was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents approximately 3.9% of the total soluble protein content of the cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 U x mg(-1) protein at 25 degrees C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of approximately 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46 degrees C after 30 min incubation; its stability is maximal in the 7.0-8.5 pH range. A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids.