RESUMO
Human interferon alpha2b (IFN-alpha2b) is a pleiotropic cytokine used for the treatment of various cancers. Antibacterial peptide CM4 is a small peptide that can strongly inhibit many kinds of bacteria, fungi, and tumor cells, but it does no harm to normal cells. Here, we describe a protein expression system for the production of IFN-alpha2b/CM4 fusion protein in insoluble form in Escherichia coli, coupled to an efficient dialysis refolding and histidine-tag purification protocol. The expressed IFN-alpha2b/CM4 fusion protein not only displays significantly improved antitumor activity, but also retains the antibacterial-antifungal activity of CM4.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Interferon-alfa/genética , Interferon-alfa/farmacologia , Animais , Anti-Infecciosos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Escherichia coli/genética , Fungos/efeitos dos fármacos , Humanos , Corpos de Inclusão , Interferon alfa-2 , Interferon-alfa/isolamento & purificação , Dobramento de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Proteínas RecombinantesRESUMO
The antimicrobial peptide CM4 is a 35-residue cationic peptide. To explore a new approach for the expression and purification of CM4 in Escherichia coli, the CM4 gene was cloned into the vector pET32a to construct an expression vector pET32a-CM4. The fusion protein Trx-CM4, purified by Ni(2+)-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. Purification of recombinant CM4 was achieved by reverse HPLC chromatography, and about 1.4 mg/l active recombinant CM4 with the purity more than 98% was obtained. The recombinant CM4 showed antimicrobial activities that were similar to synthetic one.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Fungos/efeitos dos fármacos , Vetores Genéticos , Bactérias Gram-Negativas/efeitos dos fármacos , Hidroxilamina/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Antimicrobial peptide CM4, a small cationic linear alpha-helical peptide that consists of 35 amino acids, was isolated from Bombyx mori. To improve the expression level of CM4 in Escherichia coli, tandem repeats of CM4 gene were constructed and expressed as fusion proteins (TrxA-nCM4, n=1, 2, 3,...,8) by constructing the vectors of pET32-nCM4 (n=1, 2, 3,...,8). Comparison among the expression levels of soluble fusion protein TrxA-nCM4 (n=1, 2, 3,...,8) suggested that BL21 (DE3)/pET32-3CM4 was an ideal recombinant strain for CM4 production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of CM4 was as high as 68mg/l with about 21% of fusion protein in soluble form, which was the highest yield of CM4 reported so far.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Tiorredoxinas/genética , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Bombyx/química , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/metabolismoRESUMO
Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria. Binding of LPS to the CD14+ murine macrophage cell line RAW264.7 results in pro-inflammatory cytokine secretion. In extreme cases, it leads to septic shock in vivo. Therefore, the pursuit for molecules with antiendotoxin properties is urgent. In this study, we investigated the efficacy of antibacterial peptide CM4 in binding Escherichia coli LPS in vitro. CM4 avidly bound to E. coli LPS, as proven by the limulus amoebocyte lysate assay. Furthermore, the killing activity of CM4 against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Flow cytometric analysis revealed that CM4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, the inhibition of peptide to LPS-dependent cytokine induction was analyzed. CM4 suppressed LPS-induced TNF-alpha and IL-6 mRNA expression and blocked release of TNF-alpha and NO following LPS challenge in RAW264.7 cells. Together these observations indicate that antibacterial peptide CM4 probably exerts protective actions against endotoxin shock by blocking the binding of LPS to CD14+ cells.
