1.An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.
Nuoyan ZHENG ; Xiahe HUANG ; Bojiao YIN ; Dan WANG ; Qi XIE
Protein & Cell 2012;3(12):921-928
Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
Endopeptidases
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genetics
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metabolism
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Escherichia coli
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genetics
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Plum Pox Virus
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enzymology
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genetics
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Protein Interaction Mapping
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methods
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Proteolysis
2.Asp 280 residue is important in the activity of the Escherichia coli leader peptidase.
Mee Sook SUNG ; Kwang Sook PARK
Experimental & Molecular Medicine 1999;31(2):64-69
Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.
Aspartic Acid/chemistry*
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Bacterial Outer Membrane Proteins/metabolism
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Blotting, Western
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Escherichia coli/enzymology*
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Escherichia coli/chemistry
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Micrococcal Nuclease/metabolism
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Mutagenesis, Site-Directed
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Oligonucleotides
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Protein Precursors/metabolism
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Serine Endopeptidases/metabolism*
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Serine Endopeptidases/genetics
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Serine Endopeptidases/chemistry*
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Structure-Activity Relationship
3.Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris.
Shengling YUAN ; Peng WANG ; Haoxia TAO ; Dewen ZHAN ; Yanchun WANG ; Lingchun WANG ; Chunjie LIU ; Zhaoshan ZHANG
Chinese Journal of Biotechnology 2009;25(4):526-532
Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.
Agkistrodon
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Animals
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Pichia
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genetics
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metabolism
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Recombinant Proteins
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biosynthesis
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genetics
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Serine Endopeptidases
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biosynthesis
;
genetics
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Thrombin
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biosynthesis
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genetics
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Viper Venoms
;
enzymology
4.Expression in Escherichia coli, purification and enzymatic properties of chicken aminopeptidase H.
Qingan LAI ; Shutao LIU ; Wanhua LU ; Li CHEN ; Toshihide NISHIMURA ; Pingfan RAO
Chinese Journal of Biotechnology 2008;24(3):381-386
Aminopeptidase H (APH) is an universally distributed aminoendopeptidase in the tissue of many organism. However, it is hard to investigate its mechanism underlying the catalysis and the function in cell. In this paper, the full DNA sequence of this enzyme was cloned from chicken liver, then subcloned to the vector pET22 b(+). The recombined vector was transformed into E. coli Rosetta(DE3), and the APH gene was expressed by the induction of IPTG. It was found the recombinant protein exhibited same mo lecular weight as authentic APH on SDS-PAGE analysis; the expression level increased with induction time and approached maximum of 94.7 mg/L till 6 hours, which contained 16.7% of the total protein. Moreover, this recombinant protein showed similar prop erties of subunit composition, thermal stability and optimum pH with native APH, based on the enzymatic assay, purification and analysis of enzymological properties. Therefore, it is confirmed that APH was expressed in this prokaryote system with a high-level of 1636 u/L aminopeptidase activity. These results would help to elucidate the catalysis mechanism and biological function of APH by providing enough material.
Aminopeptidases
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biosynthesis
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genetics
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Animals
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Chickens
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Endopeptidases
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biosynthesis
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genetics
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Enzyme Stability
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Escherichia coli
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genetics
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metabolism
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Recombinant Proteins
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biosynthesis
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genetics
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isolation & purification
6.Prokaryotic expression and identification of dual-fluorescence fusion proteins of small ubiquitin-like modifier and sentrin-specific protease.
Xiaohui WANG ; Jie GUO ; Jufang WANG ; Shan LI ; Lihua SUN ; Xiaoning WANG ; Jianxin LÜ
Chinese Journal of Biotechnology 2009;25(5):701-707
We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
Bacterial Proteins
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biosynthesis
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genetics
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Catalytic Domain
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Cysteine Endopeptidases
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biosynthesis
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genetics
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Endopeptidases
;
biosynthesis
;
genetics
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Escherichia coli
;
genetics
;
metabolism
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Genetic Vectors
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Green Fluorescent Proteins
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biosynthesis
;
genetics
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Humans
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Luminescent Proteins
;
biosynthesis
;
genetics
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Recombinant Fusion Proteins
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biosynthesis
;
genetics
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SUMO-1 Protein
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biosynthesis
;
genetics
7.Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme.
Xue-Ming TANG ; Zheng-Xiang WANG ; Wei-Lan SHAO ; Ji-Quan LIU ; Hui-Ying FANG ; Jian ZHUGE
Chinese Journal of Biotechnology 2002;18(6):729-734
In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.
Bacillus
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genetics
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metabolism
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Enzyme Stability
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Fermentation
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Genetic Engineering
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Metals
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pharmacology
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Recombinant Proteins
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biosynthesis
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Serine Endopeptidases
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genetics
;
isolation & purification
;
metabolism
8.A novel reporter system monitoring sortase A catalyzed protein ligation efficiency.
Jian LI ; Pengju WANG ; Yunfeng CUI ; Peijian ZOU ; Gang QIN
Chinese Journal of Biotechnology 2014;30(2):284-293
Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.
Aminoacyltransferases
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genetics
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metabolism
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Bacterial Proteins
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genetics
;
metabolism
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Biocatalysis
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Biotin
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Cysteine Endopeptidases
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genetics
;
metabolism
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli
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Genes, Reporter
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Ligation
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Mutant Proteins
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genetics
;
metabolism
9.Gene cloning and expression of serine protease SFP2 from Streptomyces fradiae var. k11.
Jiang LI ; Peng-Jun SHI ; Wang-Zhao ZHANG ; Xiao-Yu HAN ; Ling-Ling XU ; Hui-Tu ZHANG ; Bin YAO ; Yun-Liu FAN
Chinese Journal of Biotechnology 2005;21(5):782-788
Extracellular serine protease SFP2 from Streptomyces fradiae var. k11 with high feather-degrading activity was purified. The partial amino acid sequences of internal peptide of purified SFP2 were determined, and the partial gene encoding SFP2 was cloned by PCR using the degenerate primers designed according to the amino acid sequences. Complete sfp2 gene was cloned by screening the genomic DNA library of Streptomyces fradiae var. k11. The Open Reading Frame of sfp2 including pre- pro-enzyme is 924bp long (EMBL Accession number: AJ784940). The signal peptide sequence is as long as 114bp, the precursor sequence is 810bp and the mature enzyme is 576bp long, encoding 191 amino acid resides with the putative molecular weight of 19.112kD. In E. coli and Bacillus subtilis, the two sequences encoding SFP2 pro-enzyme and mature enzyme were both expressed successfully. The pro-enzyme expressed had normal biological function and its mature product had normal enzymatic activity.
Amino Acid Sequence
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Bacillus subtilis
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genetics
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metabolism
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Base Sequence
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Escherichia coli
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genetics
;
metabolism
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Molecular Sequence Data
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Serine Endopeptidases
;
genetics
;
metabolism
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Streptomyces
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enzymology
;
genetics
10.Expression and activity analysis of Enterovirus 71 3C protease in Escherichia coli.
Li CHEN ; Zhi-Jian YANG ; Zheng ZHOU ; Wei-Te CAI ; Xin-Ze TENG ; Gao-Xia ZHANG
Chinese Journal of Virology 2012;28(3):195-200
The recombinant plasmid carrying the gene encoding 3C protease of Enterovirus 71 (EV71) was constructed, the recombinant protein was then expressed and purified, the functional activity was also measured. Firstly, the 3C protease gene was inserted into pET28a vector, the constructed recombinant plasmid was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed protein was purified by affinity chromatography (Ni-NTA) and the N-terminus His-tag was cleaved by enterokinase from 3C protease. The activity of 3C protease was evaluated with fluorescent peptide substrates. It was verified by restriction analysis and sequencing that recombinant plasmid pET28a-3C was constructed correctly and functionally expressed in E. coli BL21 (DE3) resulting in the production of recombinant 3C protease with a size of 22kD. Both His-tag and non-His-tag (cleaved by enterokinase) 3C protease exhibited similar enzyme activity to 3B-3C fluorescent peptide with Km, Vmax and Kcat values of 22 microM, 434nM. Min(-1) and 0.0669 Min(-1), respectively. The optimial pH and temperature were 7.0 and 30-37 degrees C, respectively. The acquirement of recombinant purified 3C protease with high activity has paved the way of further studies on anti-viral inhibitors, structural protein assembly, vaccine development and detection methods of EV71.
Cloning, Molecular
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Cysteine Endopeptidases
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chemistry
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genetics
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metabolism
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Enterovirus A, Human
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enzymology
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genetics
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Escherichia coli
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genetics
;
metabolism
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Gene Expression
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Kinetics
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Viral Proteins
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chemistry
;
genetics
;
metabolism