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1.
Rapid Commun Mass Spectrom ; 33(12): 1067-1075, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-30900783

RESUMO

RATIONALE: LysargiNase is a novel characterized metalloprotease that can cleave the N-terminii of lysine or arginine residues. The peptides generated by LysargiNase are just mirrors to those generated by trypsin. These characteristics of LysargiNase provide a powerful tool for mass spectrometry (MS)-based proteomics research. A highly active and stable LysargiNase produced by an easy and inexpensive method could greatly benefit proteomics research. Here, we report the soluble recombinant expression, purification and acetyl modification of LysargiNase in Escherichia coli. METHODS: The coding sequence of LysargiNase with an enterokinase cleavage site at the N-terminus was inserted into plasmid pGEX-4 T-2 and transformed into E. coli BL21 (DE3). The strain was cultured in a 14-L fermenter with a working volume of 5 L. The protein expression was induced by adding isopropyl-ß-D-thiogalactoside (IPTG) to a final concentration of 1 mM. The recombinant LysargiNase was loaded onto a GSTrap and an on-column digestion was performed to remove the GST tag and was subsequently purified by chromatographic purification. In vitro acetylation of LysargiNase was performed by using acetic anhydride. The digestion efficiency and specificity of recombinant LysargiNase and acetylated LysargiNase were compared with simple protein substrate, human serum albumin (HSA), and a complex proteomic sample, yeast lysate, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: Highly soluble expression of recombinant LysargiNase was achieved by plasmid pGEX-4 T-2 in E. coli BL21 (DE3). In addition, acetylation of purified LysargiNase significantly increased its resistance to autolysis, which resulted in a more complete digestion of proteomics samples and more identified peptides and proteins by LC/MS/MS. CONCLUSIONS: In this study, we constructed a highly soluble expression system for producing recombinant LysargiNase in E. coli, which gave tremendous advantages in the downstream purification process. We also confirmed that acetyl modification can increase the stability and activity of recombinant LysargiNase. The study provided a superior way to produce this powerful tool for proteomics research.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/genética , Escherichia coli/enzimologia , Metaloproteases/química , Metaloproteases/genética , Acetilação , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/química , Escherichia coli/genética , Expressão Gênica , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Methanosarcina/enzimologia , Methanosarcina/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Proteômica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas em Tandem
2.
Sci Rep ; 8(1): 17570, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514888

RESUMO

Candida species cause cutaneous and systemic infections with a high mortality rate, especially in immunocompromised patients. The emergence of resistance to the most common antifungal drugs, also due to biofilm formation, requires the development of alternative antifungal agents. The antimicrobial peptide VLL-28, isolated from an archaeal transcription factor, shows comparable antifungal activity against 10 clinical isolates of Candida spp. Using a fluoresceinated derivative of this peptide, we found that VLL-28 binds to the surface of planktonic cells. This observation suggested that it could exert its antifungal activity by damaging the cell wall. In addition, analyses performed on biofilms via confocal microscopy revealed that VLL-28 is differentially active on all the strains tested, with C. albicans and C. parapsilosis being the most sensitive ones. Notably, VLL-28 is the first example of an archaeal antimicrobial peptide that is active towards Candida spp. Thus, this points to archaeal microorganisms as a possible reservoir of novel antifungal agents.


Assuntos
Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Arqueais/farmacologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Antifúngicos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Archaea/metabolismo , Proteínas Arqueais/isolamento & purificação , Testes de Sensibilidade Microbiana
4.
Methods Enzymol ; 606: 341-361, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30097098

RESUMO

Nitrogenase is the only known enzymatic system that converts atmospheric dinitrogen (N2) into bioavailable ammonia (NH3). The active-site cofactor responsible for this reactivity is a [(R-homocitrate)MoFe7S9C] cluster that is designated as the M-cluster. This important cofactor is assembled stepwise from a pair of [Fe4S4] clusters that become fused into a [Fe8S9C] core before additional refinements take place to complete the biosynthesis. NifB, a member of the radical S-adenosyl-l-methionine (SAM) superfamily, facilitates the conversion of the [Fe4S4] clusters (called the K-cluster) to the [Fe8S9C] core (called the L-cluster). This transformation includes a SAM-dependent carbide insertion with concomitant incorporation of an additional sulfur. While difficulties with the purification of NifB have historically prevented detailed biochemical analyses, we have developed a heterologous expression system in Escherichia coli that yields stable NifB proteins from various N2-fixing methanogenic organisms that can be used for studies. This chapter details the procedures necessary to prepare an active NifB protein. The methods used for the biochemical characterization of the SAM-dependent carbide insertion reactions are also described.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Ensaios Enzimáticos/métodos , Nitrogenase/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas Arqueais/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Compostos de Ferro/metabolismo , Methanosarcina , Nitrogenase/isolamento & purificação
5.
Methods Enzymol ; 606: 421-438, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30097101

RESUMO

Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first step is a CC bond forming reaction catalyzed by unique radical S-adenosylmethionine (SAM) enzymes. Classical radical SAM enzymes use SAM and [4Fe-4S] clusters to generate a 5'-deoxyadenynal radical and catalyze numerous reactions. Radical SAM enzymes in diphthamide biosynthesis cleave a different CS bond in SAM to generate a 3-amino-3-carboxypropyl radical and modify a histidine residue of substrate protein EF2. Here, we describe our investigations on these unique radical SAM enzymes, including the preparation, characterization, and activity assays we have developed.


Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas Arqueais/metabolismo , Ensaios Enzimáticos/métodos , Histidina/análogos & derivados , S-Adenosilmetionina/metabolismo , Alquil e Aril Transferases/isolamento & purificação , Proteínas Arqueais/isolamento & purificação , Histidina/biossíntese , Fator 2 de Elongação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Pyrococcus horikoshii
6.
Methods Enzymol ; 606: 461-483, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30097103

RESUMO

Methanogenic archaea represent a source of unique and fascinating anaerobic biochemistry that includes the involvement of many radical S-adenosyl-l-methionine (SAM) enzymes, some of which have well-established functions, while the majority have currently unknown or only partially understood functions. Here, we describe our strategy for the identification of the radical SAM enzyme that catalyzes the two methylation reactions in methanopterin biosynthesis in Methanocaldococcus jannaschii. Additionally, we describe the similar strategy carried out for the identification of the two radical SAM enzymes required for the biosynthesis of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) moiety of coenzyme F420 in M. jannaschii. This approach can be employed for future functional identification of radical SAM enzymes with currently unknown functions.


Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas Arqueais/metabolismo , Ensaios Enzimáticos/métodos , Pterinas/metabolismo , Riboflavina/análogos & derivados , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/isolamento & purificação , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Clonagem Molecular , Methanocaldococcus/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Riboflavina/biossíntese , Riboflavina/metabolismo , S-Adenosilmetionina/metabolismo
7.
Int J Biol Macromol ; 116: 817-830, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29775706

RESUMO

The present study investigates the purification and biochemical characterization of an extracellular lipase (HML) from Haloarchaea Haloferax mediterranei strain ATS1, isolated from the Sebkha (Medea, Algeria). The pure protein was obtained with ammonium sulfate precipitation (40-70%)-dialysis and UNO Q-6 FPLC, and characterized biochemically. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 45,011.09 Da. It showed optimum lipase activity at pH 7 and 60 °C. HML showed a higher specific activity on triacylglycerols with long-chains fatty acids, indicating that HML is a true lipase. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine lipase family. The Km and Vmax for HML toward olive oil were 1.01 mM and 1195 U/mg, respectively. Compared to Lipolase, HML displayed an elevated organic solvent tolerance, an outstanding stability to surfactants, oxidizing, and auxiliary agents, a considerable compatibility with various commercialized laundry detergents, and wash performance analysis revealed that it could remove oil-stains effectively. Overall, HML has a number of attractive properties that make it a potential promising candidate for the synthesis of non-aqueous peptides and detergent formulations.


Assuntos
Proteínas Arqueais/química , Haloferax mediterranei/enzimologia , Lipase/química , Solventes/química , Triglicerídeos/química , Proteínas Arqueais/isolamento & purificação , Estabilidade Enzimática , Lipase/isolamento & purificação
8.
Mol Biotechnol ; 60(6): 420-426, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29654471

RESUMO

This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.


Assuntos
Álcool Desidrogenase/metabolismo , Álcoois/metabolismo , Proteínas Arqueais/metabolismo , Halobacteriaceae/enzimologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Álcool Benzílico/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Genes Arqueais , Haloferax volcanii/genética , Temperatura Alta , Cinética , NAD/metabolismo , NADP/metabolismo , Propanóis/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
9.
Methods Mol Biol ; 1764: 307-314, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29605923

RESUMO

The archaellum assembly machinery and its filament consist of seven proteins in the crenarchaeon Sulfolobus acidocaldarius. We have so far expressed, purified, and biochemically characterized four of these archaellum subunits, namely, FlaX, FlaH, FlaI, and FlaF. FlaX, FlaH, and FlaI tightly interact and form the archaellum motor complex important for archaellum assembly and rotation. We have previously shown that FlaH forms an inner ring within a very stable FlaX ring, and therefore FlaX is believed to provide the scaffold for the assembly of the archaellum motor complex. Here we describe how to express and purify FlaX and FlaH and how the double ring structure both form can be obtained.


Assuntos
Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Membrana Celular/metabolismo , Complexos Multiproteicos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sulfolobus acidocaldarius/metabolismo , Citoplasma/metabolismo
10.
Biosci Biotechnol Biochem ; 82(8): 1327-1334, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29629656

RESUMO

In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering. This protein showed high thermal stability, with a midpoint of thermal denaturation of 79 °C in dynamic light scattering experiments. This is the first description of the carbohydrate-binding archaeal S-layer protein and its characteristics.


Assuntos
Acetilglucosamina/metabolismo , Proteínas Arqueais/metabolismo , Pyrococcus horikoshii/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Calorimetria/métodos , Cromatografia de Afinidade/métodos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Genes Arqueais , Proteínas de Fluorescência Verde/metabolismo , Temperatura Alta , Micrococcus/metabolismo , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
11.
BMC Biotechnol ; 18(1): 18, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29558934

RESUMO

BACKGROUND: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. RESULTS: Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L- 1 and 47.1 U·L- 1·h- 1 for SacPox and to 8700 U·L- 1 and 180.6 U·L- 1·h- 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. CONCLUSIONS: In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.


Assuntos
Hidrolases de Triester Fosfórico/metabolismo , Proteínas Recombinantes/isolamento & purificação , Sulfolobus acidocaldarius/enzimologia , Sulfolobus solfataricus/enzimologia , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Técnicas de Cultura Celular por Lotes/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Biodegradação Ambiental , Precipitação Química , Cromatografia em Gel/métodos , Estabilidade Enzimática , Escherichia coli/genética , Fermentação , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfolobus acidocaldarius/genética , Sulfolobus solfataricus/genética , Ultrafiltração/métodos
12.
Int J Biol Macromol ; 114: 235-243, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29551507

RESUMO

The acidophilic and thermophilic pullulanases have many potential applications in the processes of starch liquefaction and saccharification. In this study, a gene encoding an amylopullulanase from Thermofilum pendens (TPApu) was heterologously expressed in Escherichia coli. Although TPApu possessed the same continuous GH57N_Apu domain and the succeeding α-helical region as other two amylopullulanases from Staphylothermus marinus (SMApu) and Caldivirga maquilingensis (CMApu), it only showed maximal amino acid identities of 25.7-28.7% with CMApu and SMApu. The purified TPApu appeared as a single band of SDS-PAGE with a molecular mass of 65.5kDa and exhibited the maximal activity at pH3.5 and 95-100°C. TPApu had the highest catalytic efficiency towards pullulan (kcat/km, 8.79s-1mLmg-1) and α-cyclodextrin (kcat/km, 0.36s-1mM-1). In the initial stages, the ring-opening reactions of γ-cyclodextrin, 6-O-glucosyl-ß-cyclodextrin, 6-O-maltosyl-ß-cyclodextrin and the debranching reactions of 6-O-maltooctaosyl-ß-cyclodextrin were firstly catalyzed. In the subsequent reactions, a serial of maltooligosaccharides were produced. As the most acidophilic amylopullulanase among thermophilic pullulanases reported to date, TPApu preferred to debranch the DP6-12 side chains of amylopectin at pH4.5 and 100°C.


Assuntos
Proteínas Arqueais , Glicosídeo Hidrolases , Análise de Sequência de Proteína , Thermofilaceae , Proteínas Arqueais/biossíntese , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Thermofilaceae/enzimologia , Thermofilaceae/genética
13.
Methods Enzymol ; 600: 255-284, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29458762

RESUMO

Repair of DNA double-strand breaks is a critical function shared by organisms in all three domains of life. The majority of mechanistic understanding of this process has come from characterization of bacterial and eukaryotic proteins, while significantly less is known about analogous activities in the third, archaeal domain. Despite the physical resemblance of archaea to bacteria, archaeal proteins involved in break repair are remarkably similar to those used by eukaryotes. Investigating the function of the archaeal version of these proteins is, in many cases, simpler than working with eukaryotic homologs owing to their robust nature and ease of purification. In this chapter, we describe methods for purification and activity analysis for the RadA recombinase and its paralogs from the hyperthermophilic acidophilic archaeon Sulfolobus solfataricus.


Assuntos
Proteínas Arqueais/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaios Enzimáticos/métodos , Reparo de DNA por Recombinação , Sulfolobus solfataricus/genética , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria/instrumentação , Espectrofotometria/métodos , Sulfolobus solfataricus/metabolismo
14.
J Proteome Res ; 17(3): 961-977, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29301397

RESUMO

Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO_1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism.


Assuntos
Proteínas Arqueais/genética , Deleção de Genes , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/genética , Processamento de Proteína Pós-Traducional , Proteoma/genética , Proteínas Arqueais/classificação , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Adesão Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endopeptidases/deficiência , Endopeptidases/genética , Ontologia Genética , Glicosilação , Haloferax volcanii/química , Haloferax volcanii/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloproteínas/genética , Metaloproteínas/metabolismo , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/isolamento & purificação , Proteoma/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
15.
Microbiology ; 163(12): 1802-1811, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29072558

RESUMO

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor.


Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Haloferax volcanii/metabolismo , Recombinases Rec A/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Dimerização , Haloferax volcanii/química , Haloferax volcanii/genética , Ligação Proteica , Recombinases Rec A/genética , Recombinases Rec A/isolamento & purificação , Recombinases Rec A/metabolismo
16.
J Biol Chem ; 292(35): 14603-14616, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28705933

RESUMO

Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.


Assuntos
Proteínas Arqueais/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , Regulação da Expressão Gênica em Archaea , Modelos Moleculares , NADP/metabolismo , Pyrococcus furiosus/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Biocatálise , Coenzimas/química , Coenzimas/metabolismo , Cristalografia por Raios X , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/isolamento & purificação , Ferredoxinas/química , Deleção de Genes , Homeostase , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , NAD/química , NAD/metabolismo , NADP/química , Organismos Geneticamente Modificados , Oxirredução , Filogenia , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Pyrococcus furiosus/genética , Pyrococcus furiosus/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
17.
Biochem Biophys Res Commun ; 489(3): 326-331, 2017 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-28559137

RESUMO

We have exploited the self-assembling properties of archaeal-derived protein Lsmα to generate new supramolecular forms based on its stable ring-shaped heptamer. We show that engineered ring tectons incorporating cysteine sidechains on obverse faces of the Lsmα7 toroid are capable of forming paired and stacked formations. A Cys-modified construct, N10C/E61C-Lsmα, appears to organize into disulfide-mediated tube formations up to 45 nm in length. We additionally report fabrication of cage-like protein clusters through conjugation of Cu2+ to His-tagged variants of the Lsmα7 tecton. These 400 kDa protein capsules are seen as cube particles with visible pores, and are reversibly dissembled into their component ring tectons by EDTA. The ß-rich Lsmα supramolecular assemblies described are amenable to further fusion modifications, or for surface attachment, so providing potential for future applications that exploit the RNA-binding capacity of Lsm proteins, such as sensing applications.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Substâncias Macromoleculares/síntese química , Methanobacterium/química , Nanofibras/química , Engenharia de Proteínas/métodos , Proteínas Arqueais/síntese química , Proteínas Arqueais/isolamento & purificação , Substâncias Macromoleculares/química , Modelos Moleculares
19.
BMC Genomics ; 18(Suppl 2): 143, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28361671

RESUMO

BACKGROUND: The mass spectrometry based technical pipeline has provided a high-throughput, high-sensitivity and high-resolution platform for post-genomic biology. Varied models and algorithms are implemented by different tools to improve proteomics data analysis. The target-decoy searching strategy has become the most popular strategy to control false identification in peptide and protein identifications. While this strategy can estimate the false discovery rate (FDR) within a dataset, it cannot directly evaluate the false positive matches in target identifications. RESULTS: As a supplement to target-decoy strategy, the entrapment sequence method was introduced to assess the key steps of mass spectrometry data analysis process, database search engines and quality control methods. Using the entrapment sequences as the standard, we evaluated five database search engines for both the origanal scores and reprocessed scores, as well as four quality control methods in term of quantity and quality aspects. Our results showed that the latest developed search engine MS-GF+ and percolator-embeded quality control method PepDistiller performed best in all tools respectively. Combined with efficient quality control methods, the search engines can improve the low sensitivity of their original scores. Moreover, based on the entrapment sequence method, we proved that filtering the identifications separately could increase the number of identified peptides while improving the confidence level. CONCLUSION: In this study, we have proved that the entrapment sequence method could be an useful strategy to assess the key steps of the mass spectrometry data analysis process. Its applications can be extended to all steps of the common workflow, such as the protein assembling methods and data integration methods.


Assuntos
Proteínas Arqueais/isolamento & purificação , Peptídeos/isolamento & purificação , Proteômica/métodos , Ferramenta de Busca , Análise de Sequência de Proteína/métodos , Proteínas Arqueais/química , Bases de Dados de Proteínas , Conjuntos de Dados como Assunto , Humanos , Peptídeos/química , Pyrococcus furiosus/química , Controle de Qualidade , Espectrometria de Massas em Tandem
20.
Protein Expr Purif ; 129: 150-157, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27133916

RESUMO

Internal grafting of designed peptides to scaffold proteins is a valuable strategy for a variety of applications including recombinant peptide antigen construction. A peptide epitope from human papillomavirus (HPV) minor capsid protein L2 displayed on thioredoxin (Trx) has been validated preclinically as a broadly protective and low-cost alternative HPV vaccine. Focusing on thioredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus (PfTrx) as a scaffold, we have constructed a modified Pichia pastoris expression vector and used a PfTrx fusion derivative containing three tandemly repeated copies of a 19 amino acids peptide epitope from HPV-L2 for expression optimization and biochemical-immunological characterization of the Pichia-produced PfTrx-L2 antigen. We show that PfTrx-L2 is produced at high levels (up to 100 mg from a 100 ml starting culture using a multi-cycle induction protocol) and secreted into the culture medium as a highly enriched (>70% pure), non-glycosylated polypeptide that can be purified to homogeneity in a single step. Oxidation and aggregation state, thermal stability and immunogenicity of the endotoxin-free PfTrx-L2 antigen produced in P. pastoris were tested and found to be identical to those of the same antigen produced in Escherichia coli. Secretory production of endotoxin-free PfTrx-peptides in P. pastoris represents a cost- and time-effective alternative to E. coli production. Specifically designed for peptide antigens, the PfTrx-expression vector and conditions described herein are easily transferable to a variety of applications centred on the use of structurally constrained bioactive peptides as immune as well as target-specific binder reagents.


Assuntos
Proteínas Arqueais , Proteínas do Capsídeo , Papillomaviridae/genética , Pichia/metabolismo , Pyrococcus furiosus/genética , Tiorredoxinas , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Proteínas do Capsídeo/metabolismo , Temperatura Alta , Humanos , Pichia/genética , Pyrococcus furiosus/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Tiorredoxinas/metabolismo
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