RESUMO
We combined the chemical and physical methods of papain immobilization through the aldehyde groups available on oxidized bacterial cellulose (OxBC) to provide high proteolytic activity for future applications as bioactive dressing. Bacterial cellulose (BC) was obtained by the fermentation of Komagataeibacter hansenii in Hestrin-Schramm medium for 5 days, followed by purification and oxidation using NaIO4. Surface response methodology was used to optimize papain immobilization (2%, w/v) for 24 h. The independent variables: pH (3-7) and temperature (5 to 45 °C) were investigated. The mathematically validated optimal conditions of 45 °C and pH 7 had a statistical effect on the immobilization yield (IY) of papain in OxBC (52.9%). These ideal conditions were also used for papain immobilization in BC (unoxidized). The IY of 9.1% was lower than that of OxBC. OxBC-Papain and BC-Papain were investigated using thermal analysis, confocal microscopy, and diffusion testing. The OxBC support exhibited a more interactive chemical structure than the BC support, and was capable of immobilizing papain by covalent bonds (-C-NHR) and adsorption (ion exchange), with 93.3% recovered activity, 49.4% immobilization efficiency, and better thermal stability. Papain immobilized to OxBC by adsorption displayed 53% widespread papain activity. The results indicate the potential of prolonged bioactivity in debrided chronic wounds.
Assuntos
Celulose Oxidada/química , Papaína/química , Peptídeo Hidrolases/química , Pele/efeitos dos fármacos , Acetobacteraceae/enzimologia , Adsorção/efeitos dos fármacos , Celulose Oxidada/farmacologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Concentração de Íons de Hidrogênio , Oxirredução , Papaína/biossíntese , Papaína/farmacologia , Peptídeo Hidrolases/farmacologia , Pele/lesõesRESUMO
Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a(+) each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble His-tagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400mg of properly refolded propapain was obtained from 1L of bacterial culture. The expressed protein was further verified by Western blot analysis by cross-reacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease.
Assuntos
Carica/enzimologia , Precursores Enzimáticos/biossíntese , Papaína/biossíntese , Proteínas Recombinantes/biossíntese , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Escherichia coli/genética , Corpos de Inclusão/química , Cinética , Papaína/genética , Papaína/isolamento & purificação , Reação em Cadeia da Polimerase , Engenharia de Proteínas/métodos , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Alkylation of a cysteine residue in papain with a pyridoxamine (PX) cofactor was carried out. The resulting semisynthetic enzyme (papain-PX) has no detectable protease activity but has the ability to catalyze enantioselective reductive amination of alpha-keto acids. The conjugate was characterized by ion-exchange chromatography, and the optimal reaction conditions were found. We report that papain-PX reductively aminates the alkyl side chain of functionalized alpha-keto acids to give the respective alpha-amino acids with high enantioselectivities, greater than 70%. Based on these studies, we propose a new model for the catalytic activity of the semisynthetic enzyme with Interchem software. The results of the study demonstrate the effectiveness of the modified enzyme and its potential for engineering new catalytic specificity.
Assuntos
Cetoácidos/metabolismo , Papaína/metabolismo , Aminação , Catálise , Cromatografia por Troca Iônica , Cinética , Modelos Moleculares , Papaína/biossíntese , Papaína/química , Engenharia de Proteínas , Piridoxamina/química , Software , Estereoisomerismo , Especificidade por SubstratoRESUMO
We have transferred the cloned papain genetic information into an expression vector (pT7-7) regulated by the T7-promoter and have obtained in vitro expression as well as expression in Escherichia coli. In Western blots the proteins produced are immunologically recognizable as papain. Multiple forms of specific but differing sizes are detected, suggesting either that initiation can occur at more than one of the upstream methionines, or that the enzyme is processed after synthesis.
Assuntos
Escherichia coli/genética , Papaína/biossíntese , DNA Recombinante , Precursores Enzimáticos/biossíntese , Escherichia coli/enzimologia , Iniciação Traducional da Cadeia Peptídica , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The baculovirus/insect cell system has been remarkably successful in yielding high levels of synthesis of many proteins which have been difficult to synthesize in other host/vector systems. The system is also capable of secreting heterologous proteins, but with generally low efficiency. We have increased the efficiency of secretion of the system by using signal peptides of insect origin to direct the secretion of a foreign protein. The precursor of the plant cysteine protease papain (propapain) has been used as a report enzyme to compare secretion efficiency. Insect cells infected with a baculovirus recombined with the gene encoding propapain fused to the sequence encoding the honeybee melittin signal peptide secreted over five times more papain precursor than the wild-type prepropapain which used the plant signal peptide. Based on these results, we have assembled pVT-Bac, an Autographa californica nuclear polyhedrosis virus transfer vector that may enhance secretion of other foreign proteins from insect cells. The vector incorporates a number of features: phage f1 ori to facilitate site-directed mutagenesis, the strong polyhedrin promoter upstream from the melittin signal peptide-encoding sequence, and eight unique restriction sites to facilitate fusion of heterologous genes.
Assuntos
Meliteno/genética , Papaína/genética , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Insetos , Cinética , Meliteno/biossíntese , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Papaína/biossíntese , Plantas/enzimologia , Plantas/genética , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , TransfecçãoRESUMO
A 1048-bp gene coding for prepropapain was assembled from chemically synthesized oligodeoxyribonucleotides and cloned into a variety of Escherichia coli expression plasmids. We observed loss of plasmid when the preproP gene was expressed in E. coli either as the native precursor or fused at the C terminus of the first 592 amino acids (aa) of beta-galactosidase (beta Gal). Deletion of the putative 26-aa signal peptide (pre-region) increased plasmid stability. The level of maintenance for the different plasmid constructs correlated with the level of expression detected by immunoblotting. Constitutive expression of the beta Gal-propapain fusion generated insoluble granules in a protease-deficient E. coli host. The fusion protein was easily purified to near homogeneity by differential solubilization of the granules.
Assuntos
Escherichia coli/genética , Regulação da Expressão Gênica , Genes Sintéticos , Papaína/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , DNA , Dados de Sequência Molecular , Mutação , Papaína/biossíntese , Plasmídeos , Precursores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Viral proteases are essential for pathogenesis and virulence of severe acute respiratory syndrome coronavirus (SARS-CoV). Little information is available on SARS-CoV papain-like protease 2 (PLP2), and development of inhibitors against PLP2 is attractive for antiviral therapy. Here, we report the characterization of SARS-CoV PLP2 (from residues 1414 to 1858) purified from baculovirus-infected insect cells. We demonstrate that SARS-CoV PLP2 by itself differentially cleaves between the amino acids Gly180 and Ala181, Gly818 and Ala819, and Gly2740 and Lys2741 of the viral polypeptide pp1a, as determined by reversed-phase high-performance liquid chromatography analysis coupled with mass spectrometry. This protease is especially selective for the P1, P4, and P6 sites of the substrate. The study demonstrates, for the first time among coronaviral PLPs, that the reaction mechanism of SARS-CoV PLP2 is characteristic of papain and compatible with the involvement of the catalytic dyad (Cys)-S(-)/(His)-Im(+)H ion pair. With a fluorogenic inhibitor-screening platform, we show that zinc ion and its conjugates potently inhibit the enzymatic activity of SARS-CoV PLP2. In addition, we provided evidence for evolutionary reclassification of SARS-CoV. The results provide important insights into the biochemical properties of the coronaviral PLP family and a promising therapeutic way to fight SARS-CoV.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Papaína/genética , Papaína/isolamento & purificação , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Proteases Semelhantes à Papaína de Coronavírus , Coronavirus Bovino/enzimologia , Inibidores de Cisteína Proteinase/síntese química , Hidrólise , Concentração Inibidora 50 , Cinética , Dados de Sequência Molecular , Vírus da Hepatite Murina/enzimologia , Papaína/antagonistas & inibidores , Papaína/biossíntese , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Especificidade por Substrato , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/biossíntese , Zinco/farmacologiaRESUMO
The S2 subsite specificity of the plant protease papain has been altered to resemble that of mammalian cathepsin B by site-directed mutagenesis. On the basis of amino acid sequence alignments for papain and cathepsin B, a double mutant (Val133Ala/Ser205Glu) was produced where Val133 and Ser205 are replaced by Ala and Glu, respectively, as well as a triple mutant (Val133Ala/Val157Gly/Ser205Glu), where Val157 is also replaced by Gly. Three synthetic substrates were used for the kinetic characterization of the mutants, as well as wild-type papain and cathepsin B: CBZ-Phe-Arg-MCA, CBZ-Arg-Arg-MCA, and CBZ-Cit-Arg-MCA. The ratio of kcat/KM obtained by using CBZ-Phe-Arg-MCA as substrate over that obtained with CBZ-Arg-Arg-MCA is 8.0 for the Val133Ala/Ser205Glu variant, while the equivalent values for wild-type papain and cathepsin B are 904 and 3.6, respectively. This change in specificity has been achieved by replacing only two amino acids out of a total of 212 in papain and with little loss in overall enzyme activity. However, further replacement of Val157 by Gly as in Val133Ala/Val157Gly/Ser205Glu causes an important decrease in activity, although the enzyme still displays a cathepsin B like substrate specificity. In addition, the pH dependence of activity for the Val133Ala/Ser205Glu variant compares well with that of cathepsin B. In particular, the activity toward CBZ-Arg-Arg-MCA is modulated by a group with a pKa of 5.51, a behavior that is also encountered in the case of cathepsin B but is absent with papain.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Mutagênese Sítio-Dirigida , Papaína/genética , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , Humanos , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Dados de Sequência Molecular , Papaína/biossíntese , Ratos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication. In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for PLP-1. We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein. With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates. Subsequent characterization of the recombinant PLP-1 revealed that in vitro trans cleavage is more efficient at 22 degrees C than at higher temperatures. Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa. In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed. The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans. In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage in cis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme.
Assuntos
Coronavirus/genética , Papaína/genética , Proteínas Recombinantes/genética , Animais , Sequência de Bases , Clonagem Molecular , Proteases Semelhantes à Papaína de Coronavírus , Camundongos , Dados de Sequência Molecular , Papaína/biossíntese , Proteínas Recombinantes/biossíntese , Especificidade por SubstratoRESUMO
The synthetic gene coding for the precursor of the cysteine protease papain (EC 3.4.22.2) has been expressed using the baculovirus/insect cell system. The prepropapain gene was cloned into the transfer vector IpDC125 behind the polyhedrin promoter. The recombinant construct was then incorporated by homologous recombination into the Autographa californiaca nuclear polyhedrosis virus genome. The host Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus secrete an enzymatically inactive N-glycosylated papain precursor. This zymogen could be activated in vitro to yield about 400 nmol of active papain per liter of culture. The recombinant active mature papain was enzymatically indistinguishable from natural papain but the precursor was not processed to the same amino acid residue. The insect cells also accumulated prepropapain and glycosylated propapain intracellularly. This accumulation was an indication that there are rate-limiting steps in the secretion of proteins from insect cells in this expression system. Characterization of mutants of the precursor has shown that entry into the secretory pathway and addition of carbohydrate are prerequisite conditions for the production and secretion of functional propapain.
Assuntos
Precursores Enzimáticos/genética , Papaína/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Precursores Enzimáticos/biossíntese , Genes Sintéticos , Glicosilação , Vírus de Insetos/genética , Dados de Sequência Molecular , Mariposas , Mutação , Sondas de Oligonucleotídeos , Papaína/biossíntese , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossínteseRESUMO
A microbial expression system for the study of the cysteine protease papain has been developed as a more useful alternative to the insect cell/baculovirus expression system we have previously used. A synthetic papain precursor (propapain) gene was expressed in the yeast Saccharomyces cerevisiae under the control of the alpha-factor promoter. Efficient expression required fusion of the propapain sequence with the yeast alpha-factor prepro region and a yeast host cell defective in the synthesis of vacuolar proteases. Surprisingly, the glycosylated form of the inactive papain precursor is not secreted, but accumulates within the yeast cell. Complete conversion of the intracellular zymogen into active mature papain could be achieved in vitro. Purified recombinant papain produced by the yeast system has kinetic characteristics similar to those of the natural enzyme. An advantage of the yeast expression system over the baculovirus/insect cell system is that we can perform mutagenesis and screening of papain mutants very efficiently. We have set up a 'one-tube' screening procedure for the simultaneous characterization of numerous mutants of the papain precursor. Yeast cells are grown and lysed in microtiter plate wells and the released papain precursor is then activated to mature papain. This assay allows easy discrimination between proteins with close to wild type properties and proteins that are not functional. We have applied this assay to investigate the spectrum of amino acids which are tolerated at Asn175 of papain using two independently derived libraries of mutants at this position. Many amino acid substitutions at this position are not accepted; only the reintroduction of Asn restored normal function.
Assuntos
Papaína/genética , Precursores de Proteínas/biossíntese , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biblioteca Gênica , Glicosilação , Fator de Acasalamento , Dados de Sequência Molecular , Papaína/biossíntese , Peptídeos/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-TraducionalRESUMO
Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteinases encoded within the first open reading frame (ORF 1a) of the replicase gene. The more 5' of these domains, the leader papain-like proteinase, is responsible for the cleavage of the amino terminal protein, p28. The core of this proteinase domain was defined to between amino acids 1084 and 1316 from the beginning of ORF 1a. Through the use of deletion analysis coupled with in vitro expression, we studied the role of the coding region between p28 and the leader papain-like proteinase on the cleavage of p28 itself. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels. Reduced p28 production resulting from a 0.4-kb deletion positioned between p28 and the proteinase domain suggests an involvement of this region in catalytic processing. Some mutants displayed cleavage patterns indicative of a second cleavage site. Interestingly, this new cleavage site identified in vitro maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59-infected cells. Mutagenesis of the catalytic His1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like proteinase encoded in ORF 1a.
Assuntos
Vírus da Hepatite Murina/enzimologia , Papaína/biossíntese , Papaína/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteases Semelhantes à Papaína de Coronavírus , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Epitopos/análise , Genoma Viral , Histidina , Camundongos , Dados de Sequência Molecular , Vírus da Hepatite Murina/genética , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Papaína/isolamento & purificação , Plasmídeos , Reação em Cadeia da Polimerase , Fases de Leitura , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Mapeamento por Restrição , Deleção de SequênciaRESUMO
For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.