精氨酸脱亚胺酶在大肠杆菌中的分泌型表达

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arc A克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢB中,经鉴定得到重组质粒pBAD-ADI。将重组质粒转化大肠杆菌TOP10F'后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002%(w/v),25℃下诱导5 h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同)。采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL。SDS-PAGE分析显示,重组蛋白大小约为46 kD。     

精氨酸脱亚胺酶发酵过程特性研究

精氨酸脱亚胺酶有较好的体内体外肿瘤生长抑制作用。通过对粪肠球菌（Enterococcus faecalis）NJ402菌株产精氨酸脱亚胺酶的发酵特性的研究,建立起代谢物的过程变化与精氨酸脱亚胺酶产生机理的内在联系。NJ402菌株生长过程中碳源物质代谢产生乳酸导致发酵体系pH的下降,而培养基中L-精氨酸的脱亚胺作用有利于发酵体系pH的稳定和菌体生长。进一步的研究表明,低pH生长环境有利于NJ402菌株产精氨酸脱亚胺酶,且精氨酸脱亚胺酶的产生与能量代谢无关。NJ402菌株产精氨酸脱亚胺酶受底物L-精氨酸的诱导,但该诱导作用受菌体生长体系pH的调控,即精氨酸脱亚胺酶的产生是低pH生长环境与L－精氨酸共同作用的结果。     

Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, its application as anticancer drug is hampered by its poor stability under physiological conditions in the bloodstream. Commonly, random PEGylation is being used for increasing the stability of ADI and in turn the improved half-life. However, the traditional random PEGylation usually leads to poor PEGylation efficiency due to the limited number of Lys on the protein surface. To boost the PEGylation efficiency and enhance the stability of ADI further, surface engineering of PpADI (an ADI from Pseudomonas plecoglossicida) to increase the suitable PEGylation sites was carried out. A new in silico approach for increasing the PEGylation sites was developed. The validation of this approach was performed on previously identified PpADI variant M31 to increase potential PEGylation sites. Four Arg residues on the surface of PpADI M31 were selected through three criteria and subsequently substituted to Lys, aiming for providing primary amines for PEGylation. Two out of the four substitutions (R299K and R382K) enhanced the stability of PEGylated PpADI in human serum. The average numbers of PEGylation sites were increased from ~12 (tetrameric PpADI M31, starting point) to ~20 (tetrameric PpADI M36, final variant). Importantly, the PEGylated PpADI M36 after PEGylation exhibited significantly improved T_m values (M31: 40°C; M36: 40°C; polyethylene glycol [PEG]-M31: 54°C; PEG-M36: 64°C) and half-life in human serum (M31: 1.9 days; M36: 2.0 days; PEG-M31: 3.2 days; PEG-M36: 4.8 days). These proved that surface engineering is an effective approach to increase the PEGylation efficiency which therefore enhances the stability of therapeutic enzymes. Furthermore, the PEGylated PpADI M36 represents a highly attractive candidate for the treatment of arginine-auxotrophic tumors.     

RNA interference of cofilin in Chinese hamster ovary cells improves recombinant protein productivity

RNA interference (RNAi) has been recently applied to improve the yield and quality of recombinant proteins produced in Chinese hamster ovary (CHO) cells, the most commonly used mammalian cell line for production of complex biopharmaceuticals. Proteomic profiling of CHO cells undergoing gene amplification identified cofilin, a key regulatory protein of actin cytoskeletal dynamics, as a cellular target for genetic engineering studies. Transient reduction of cofilin by small interfering RNA (siRNA) enhanced specific productivity in recombinant CHO cells by up to 80%. CHO cell lines expressing cofilin-specific short hairpin RNA (shRNA) vectors showed up to a 65% increase in specific productivity. These results suggest that modulation of cofilin, and its regulatory pathways, may be a new approach to enhance recombinant protein productivity in CHO cells.     

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.     

Development of a highly sensitive fluorescence probe for peptidyl arginine deiminase (PAD) activity

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer’s disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.     

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

Targeting specific genes for RNA interference is crucial to the development of strong resistance to rice stripe virus

Rice stripe virus (RSV) has a serious negative effect on rice production in temperate regions of East Asia. Focusing on the putative importance of the selection of target sequences for RNA interference (RNAi), we analysed the effects of potential target sequences in each of the coding genes in the RSV genome, using transgenic rice plants that expressed a set of inverted-repeat (IR) constructs. The reactions of inoculated transgenic T(1) plants to RSV were divided subjectively into three classes, namely highly resistant, moderately resistant and lacking enhanced resistance to RSV, even though plants that harboured any constructs accumulated transgene-specific siRNAs prior to inoculation with RSV. Transgenic plants that harboured IR constructs specific for the gene for pC3, which encodes nucleocapsid protein, and for pC4, which encodes a viral movement protein, were immune to infection by RSV and were more resistant to infection than the natural resistant cultivars that have been used to control the disease in the field. By contrast, the IR construct specific for the gene for pC2, which encodes a glycoprotein of unknown function, and for p4, which encodes a major non-structural protein of unknown function, did not result in resistance. Our results indicate that not all RNAi constructs against viral RNAs are equally effective in preventing RSV infection and that it is important to identify the viral 'Achilles heel' for RNAi attack in the engineering of plants.     

Purification,immobilization, and biochemical characterization of l‐arginine deiminase from thermophilic Aspergillus fumigatus KJ434941: Anticancer activity in vitro

l ‐Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G₁ phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1‐fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG‐ADI, and Dex‐ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG‐ADI displays twofold resistance to thermal denaturation (t_1/2 13.9 h), than free ADI (t_1/2 6.9 h), while at 70°C, the thermal stability of PEG‐ADI was increased by 1.7‐fold, with similar stability to Dex‐ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG‐ADI and Dex‐ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG‐ADI, and Dex‐AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP‐G2, and MCF7, in vitro. The free and PEG‐ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex‐ADI. The free ADI had IC₅₀ value 22.0, 16.6, and 13.9 U/mL, while Dex‐ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG‐2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG‐2 was increased by five‐, three‐, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti‐ADI immunoglobulins in vivo. The in vivo half‐life time of free ADI, PEG‐ADI, and Dex‐ADI was 29.7, 91.1, 59.6 h, respectively. The present findings explored a novel thermostable, less antigenic ADI from thermophilic A. fumigatus, with further molecular and crystallographic analyses, this enzyme will be a powerful candidate for clinical trials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:396–405, 2015     

Glutamine synthetase and arginine inhibition of nitrate reductase activity in Anabaena cycadeae

Wild-type Anabaena cycadeae with normal glutamine synthetase (GS) activity utilized arginine as sole N source whereas a mutant strain lacking GS activity did not. Nitrate reductase (NR) activity, higher in the mutant strain than the wild-type strain, was inhibited by arginine though arginine-dependent NH ₄ ⁺ generation was higher in the mutant strain than in the wild-type. This suggests that (1) NR activity is NO _inf3 ^sup- -inducible and arginine-repressible; and (2) while GS activity is required for the assimilation of arginine as sole N-source, it is not required for arginine inhibition of NR activity.S. Singh was with the Department of Biochemistry, North-Eastern Hill University, Shillong-793014, India, and is now with P.S. Bisen at the Department of Microbiology, Barkatullah University, Bhopal-462026, India     

Applications of RNA interference high-throughput screening technology in cancer biology and virology

RNA interference (RNAi) is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi high-throughput screening (HTS) has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.     

The response of mammalian cells to double-stranded RNA

Double-stranded RNA (dsRNA) has long been recognized as a central component of the interferon (IFN) system. It was originally characterized as a key mediator of IFN induction in response to virus infection. Subsequently, it was identified as a prime activator of the antiviral response. In recent years the discovery of the RNA interference (RNAi) pathway in mammals has renewed interest in dsRNA-mediated cellular responses. This has coincided with the identification of key components of the IFN induction pathway. Here, we present an overview of the current knowledge of dsRNA-mediated pathways in mammalian cells and introduce a link between these pathways and application of RNAi.     

Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel)

In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS‐specific inhibitor (L‐methionine S‐sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS‐c and BdGS‐m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS‐c) and mitochondria (BdGS‐m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS‐c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS‐c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS‐c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS‐c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management.     

RNA interference in immune cells by use of osmotic delivery of siRNA

Delivery of siRNA to immune cells has been one of the major obstacles to widespread application of RNAi in the immunology field. Here, we report that osmotic delivery of siRNA can be used to silence genes in macrophage RAW264.7 without incurring either cytotoxic or immunomodulatory activity. We also showed usefulness of the osmotic delivery in other types of cells including T cell DO11.10. By repeated osmotic delivery of siRNA, long-term gene silencing was readily achieved. When TLR4 was disrupted in RAW264.7 cells for 48 h and the cells were stimulated with the TLR4 ligand LPS, a significant decrease in TNFalpha production was observed. DNA microarray-based gene expression profile analysis showed that gene silencing by osmotic delivery of siRNA was target-specific and the delivery method itself had little influence on overall gene expression.     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.