RESUMEN
An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) Escherichia coli strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against Staphylococcus aureus, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae bacteria clinical isolates at a lower minimum inhibition concentration level (10-33 µM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50-100 µM). KEY POINTS: ⢠The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker. ⢠The peptide was expressed with the carrier proteins SmbP and CusF3H+. ⢠The hybrid peptide shows antibacterial potency against clinical bacterial isolates.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Catelicidinas/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Bacterias , Staphylococcus aureus , Escherichia coli/genética , Proteínas Portadoras/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
The bacterium Escherichia coli is still considered the first option as a microbial cell factory for recombinant protein production, and affinity chromatography is by far the preferred technique for initial purification after protein expression and cell lysis. In this chapter, we describe the methodology to express and purify recombinant proteins in E. coli tagged with the first two metal-binding proteins proposed as fusion partners. They are the small metal-binding protein SmbP and a mutant of the copper resistance protein CusF3H+. There are several advantages of using them as protein tags: they prevent the formation of inclusion bodies by increasing solubility of the target proteins, they enable purification by immobilized metal-affinity chromatography using Ni(II) ions with high purity, and because of their low molecular weights, excellent final yields are obtained for the target proteins after cleavage and removal of the protein tag. Here we also describe the protocol for the production of proteins in the periplasm of E. coli tagged with two SmbP variants that include the PelB or the TorA signal sequences for transport via the Sec or the Tat pathway, respectively. Based on these methods, we consider CusF3H+ and SmbP excellent alternatives as fusion proteins for the production of recombinant proteins in E. coli.
Asunto(s)
Cromatografía de Afinidad , Proteínas Transportadoras de Cobre , Proteínas de Escherichia coli , Escherichia coli/química , Níquel/química , Periplasma/química , Proteínas Transportadoras de Cobre/química , Proteínas Transportadoras de Cobre/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Periplasma/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
BACKGROUND: The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. OBJECTIVES: Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6.9 kDa) in Escherichia coli. METHODS: The recombinant VpDef (rVpDef) peptide was expressed as a translational fusion with CusF3H+ and SmbP in Escherichia coli SHuffle under different small-scale culture conditions. The best conditions were applied to 1-liter cultures, with subsequent purification of the recombinant protein through IMAC chromatography. The recombinant protein was digested using enterokinase to liberate the peptide from the fusion protein, and a second IMAC chromatography step removed the fusion protein. The purified peptide was tested against two Gram-positive and two Gram-negative bacteria. RESULTS: The use either of CusF3H+ or of SmbP results in recombinant proteins that are found in the soluble fraction of the bacterial lysate; these recombinant proteins are easily purified through IMAC chromatography, and rVpDef is readily separated following enterokinase treatment. The purified rVpDef peptide exhibits antimicrobial properties against both Gram-positive and Gram-negative. CONCLUSION: Use of the fusion proteins CusF3H+ and SmbP results in production of a soluble recombinant protein containing the antimicrobial peptide rVpDef that is correctly folded and that retains its antimicrobial properties once purified.
Asunto(s)
Proteínas Transportadoras de Cobre , Defensinas , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión , Proteínas Transportadoras de Cobre/biosíntesis , Proteínas Transportadoras de Cobre/química , Proteínas Transportadoras de Cobre/genética , Proteínas Transportadoras de Cobre/aislamiento & purificación , Defensinas/biosíntesis , Defensinas/química , Defensinas/genética , Defensinas/aislamiento & purificación , Escherichia coli/genética , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography.
RESUMEN
We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.
Asunto(s)
Proteínas Bacterianas/genética , Clonación Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas Transportadoras de Cobre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Periplasma/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteína Fluorescente RojaRESUMEN
Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas Bacterianas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Transporte de Catión , Proteínas de Escherichia coli , Escherichia coli , Synechocystis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/aislamiento & purificación , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/aislamiento & purificación , Proteínas Transportadoras de Cobre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Synechocystis/metabolismoRESUMEN
Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography.
RESUMEN
Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.
Asunto(s)
Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/aislamiento & purificación , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/química , Proteínas Transportadoras de Cobre , Escherichia coli/genética , Proteínas de Escherichia coli/química , Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Histidina/química , Metales/química , Proteínas Recombinantes de Fusión/genética , SolubilidadRESUMEN
The periplasmic protein CusF acts as a metallochaperone to mediate Cu resistance in Escherichia coli. CusF does not contain cysteine residues and barely binds to divalent cations. Here, we addressed effects of cysteine-substitution mutant (named as mCusF) of CusF on zinc/cadmium (Zn/Cd) accumulation and resistance. We targeted mCusF to different subcellular compartments in Arabidopsis. We found that plants expressing vacuole-targeted mCusF were more resistant to excess Zn than WT and plants with cell wall-targeted or cytoplasmic mCusF. Under long-term exposure to excess Zn, all transgenic lines accumulated more Zn (up to 2.3-fold) in shoots than the untransformed plants. Importantly, plants with cytoplasmic mCusF showed higher efficiency of Zn translocation from root to shoot than plants with secretory pathway-targeted-mCusF. Furthermore, the transgenic lines exhibited enhanced resistance to Cd and significant increase in root-to-shoot Cd translocation. We also found all transgenic plants greatly improved manganese (Mn) and iron (Fe) homeostasis under Cd exposure. Our results demonstrate heterologous expression of mCusF could be used to engineer a new phytoremediation strategy for Zn/Cd and our finding also deepen our insights into mechanistic basis for relieving Cd toxicity in plants through proper root/shoot partitioning mechanism and homeostatic accumulation of Mn and Fe.
Asunto(s)
Arabidopsis/metabolismo , Cadmio/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Escherichia coli/genética , Plantas Modificadas Genéticamente/metabolismo , Zinc/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Biodegradación Ambiental , Proteínas de Transporte de Catión/metabolismo , Proteínas Transportadoras de Cobre , Proteínas de Escherichia coli/metabolismo , Ingeniería Genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Estrés FisiológicoRESUMEN
Copper (Cu) is an important environmental pollutant that exerts harmful effects on all living organisms when in excess. In an effort to remove this toxin in situ, a bacterial Cu-binding protein gene CusF was engineered to target CusF for secretion to the cell wall and vacuoles and for accumulation in the cytoplasm. Analysis of transgenic Arabidopsis plants showed that CusF was functionally active and that plants expressing cell wall- (CusFcw transgenic lines) or vacuole-targeted CusF (CusFvac transgenic lines) were more resistant to Cu excess than untransformed plants and plants with cytoplasmic CusF (CusFcyto transgenic lines). Under short-term (48 h) exposure to Cu excess, CusFcw transgenic lines showed up to 2-fold increased Cu accumulation in roots compared with the untransformed plants; however, CusFcyto lines and the wild-type plants had similar Cu concentrations in both roots and shoots. Under long-term (40 d) exposure to Cu excess, all transgenic lines accumulated more Cu (up to 3-fold) in roots than the untransformed plants, whereas only CusFcyto lines showed a marked increase (â¼3-fold of the wild-type plants) of Cu accumulation in shoots. In addition, expression of CusF in the cytosol dramatically enhanced Cu transport from roots to shoots when compared with plants with secretory pathway-targeted CusF. Our results demonstrate the feasibility of Cu tolerance and accumulation by engineering Cu-binding proteins targetable to subcellular compartments and provide new insights into the multifaceted mechanisms of Cu partitioning between roots and shoots.