Assuntos
Cryptomeria , Alérgenos , Antígenos de Plantas , Giberelinas , Humanos , Proteínas de Plantas , PólenRESUMO
Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and (1)H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris.
Assuntos
Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Clonagem Molecular , Pichia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Solanum tuberosum/genética , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Clonagem Molecular/métodos , Fungos/efeitos dos fármacos , Humanos , Micoses/tratamento farmacológico , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Transformação GenéticaRESUMO
Snakin-1 (SN-1) is a cysteine-rich plant antimicrobial peptide and the first purified member of the snakin family. SN-1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN-1 in Escherichia coli by a previously developed coexpression method using an aggregation-prone partner protein. Our goal was to increase the productivity of SN-1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN-1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN-1, the identity of which was verified by MALDI-TOF MS and NMR studies. The purified recombinant SN-1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation-prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN-1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520-1528, 2017.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Proteínas de Plantas/biossíntese , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Anti-Infecciosos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Clonagem Molecular , Cisteína/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Humanos , Proteínas de Plantas/genética , Proteínas de Plantas/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêuticoRESUMO
Trans-isoferulic acid was isolated from the carbon tetrachloride soluble fraction of a methanol extract of the rhizomes of Curcuma longa (Zingiberaceae). The structure of the isolated compound was elucidated by comprehensive analysis of spectroscopic data. This is the first report of its occurrence from this plant.
El ácido trans-isoferulico fue aislado de la fracción soluble en tetracloruro de carbono del extracto metanólico de los rizomas de Curcuma Longa (Zingeraceae). La estructura del compuesto aislado fue elucidada por análisis de los datos espectroscópicos. Este es el primer reporte de su presencia en esta planta.