RESUMO
Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an α helix in the complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.
Assuntos
Proteínas de Protozoários/química , RNA de Protozoário/química , RNA/química , Ribonucleoproteínas/química , Telomerase/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA/genética , RNA/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Homologia de Sequência de Aminoácidos , Telomerase/genética , Telomerase/metabolismo , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/genéticaRESUMO
The α-synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson's disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α-synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme-based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over-expressers, knock-outs) suggested a concentration dependence of α-synuclein on calcium/depolarization-dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α-synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α-synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno-gold and transmission electron microscopy (TEM) detected exogenous fibrillated α-synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno-gold TEM were consistent with SN internalization of α-synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α-synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α-synuclein or extrasynaptic exposure to fibrillated α-synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies. © 2017 Wiley Periodicals, Inc.
Assuntos
Ácido Glutâmico/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/farmacologia , Animais , Humanos , Camundongos , Doença de ParkinsonRESUMO
Human chemokine (C-C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags-Trx, GST, MBP, NusA, His8, PDI, and PDIb'a'-for their ability to allow the soluble overexpression of hCCL2. Three tags-MBP, His8, and PDI-solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18 °C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor.
Assuntos
Quimiocina CCL2/genética , Escherichia coli/genética , Expressão Gênica , Proteínas Ligantes de Maltose/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Linhagem Celular , Quimiocina CCL2/metabolismo , Escherichia coli/metabolismo , Ordem dos Genes , Humanos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics.
Assuntos
Eritropoetina/isolamento & purificação , Eritropoetina/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proliferação de Células , Clonagem Molecular , Eritropoetina/química , Eritropoetina/genética , Escherichia coli/genética , Humanos , Proteínas Ligantes de Maltose/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , SolubilidadeRESUMO
This protocol describes a method to purify SOD1 in Saccharomyces cerevisiae to characterize using ICP-MS and AFM, to agitate and fibrillate for aggregation of SOD1. The human SOD1 (hSOD1) is a 32-kDa homodimer, with one copper- and one zinc-binding site per 153-amino acid subunit. Misfolded protein aggregates are often correlated with diseases known as amyloidosis, including ALS, Alzheimer's, Parkinson's, and prion disease (Valentine and Hart, Proc Natl Acad Sci USA 100: 3617-3622, 2003; Tanzi and Bertram, Cell 120: 545-555, 2005; Soto and Pritzkow, Nat Neurosci 21:1332-1340, 2018; Sarafian et al., J Neurosci Res 95:1871-1887, 2017). Proteinaceous aggregates containing hSOD1 have frequently been found in the spinal cords of ALS patients.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase/metabolismo , Microscopia de Força Atômica , Proteólise , Agregados Proteicos , Espectrometria de Massas , MutaçãoRESUMO
Wild-type human SOD1 forms a highly conserved intra-molecular disulfide bond between C57-C146, and in its native state is greatly stabilized by binding one copper and one zinc atom per monomer rendering the protein dimeric. Loss of copper extinguishes dismutase activity and destabilizes the protein, increasing accessibility of the disulfide with monomerization accompanying disulfide reduction. A further pair of free thiols exist at C6 and C111 distant from metal binding sites, raising the question of their function. Here we investigate their role in misfolding of SOD1 along a pathway that leads to formation of amyloid fibrils. We present the seeding reaction of a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) to exclude variables caused by these free cysteines. Completely reduced fibril seeds decreasing the kinetic barrier to cleave the highly conserved intramolecular disulfide bond, and accelerating SOD1 reduction and initiation of fibrillation. Presence or absence of the pair of free thiols affects kinetics of fibrillation. Previously, we showed full maturation with both Cu and Zn prevents this behavior while lack of Cu renders sensitivity to fibrillation, with presence of the native disulfide bond modulating this propensity much more strongly than presence of Zn or dimerization. Here we further investigate the role of reduction of the native C57-C146 disulfide bond in fibrillation of wild-type hSOD1, firstly through removal of free thiols by paired mutations C6A, C111S (AS-SOD1), and secondly in seeded fibrillation reactions modulated by reductant tris (2-carboxyethyl) phosphine (TCEP). Fibrillation of AS-SOD1 was dependent upon disulfide reduction and showed classic lag and exponential growth phases compared with wild-type hSOD1 whose fibrillation trajectories were typically somewhat perturbed. Electron microscopy showed that AS-SOD1 formed classic fibrils while wild-type fibrillation reactions showed the presence of smaller "sausage-like" oligomers in addition to fibrils, highlighting the potential for mixed disulfides involving C6/C111 to disrupt efficient fibrillation. Seeding by addition of sonicated fibrils lowered the TCEP concentration needed for fibrillation in both wild-type and AS-SOD1 providing evidence for template-driven structural disturbance that elevated susceptibility to reduction and thus propensity to fibrillate.
RESUMO
The syndecan transmembrane proteoglycans are involved in the organization of the actin cytoskeleton and have important roles as cell surface receptors during cell-matrix interactions. We have shown that the syndecan-4 cytoplasmic domain (4L) forms oligomeric complexes that bind to and stimulate PKCalpha activity in the presence of PtdIns(4,5)P2, emphasizing the importance of multimerization in the regulation of PKCalpha activation. Oligomerization of the cytoplasmic domain of syndecan-4 is regulated either positively by PtdIns(4,5)P2 or negatively by phosphorylation of serine 183. Phosphorylation results in reduced PKCalpha activity by inhibiting PtdIns(4,5)P2-dependent oligomerization of the syndecan-4 cytoplasmic domain. Data from NMR and gel-filtration chromatography show that the phosphorylated cytoplasmic domain (p-4L) exists as a dimer, similar to 4L, but not as higher-order oligomers. NMR analysis showed that the overall conformation of p-4L is a compact intertwined dimer with an unusually symmetric clamp shape, and its molecular surface is mostly positively charged. The two parallel strands form a cavity in the center of the dimeric twist. An especially marked effect of phosphorylation of the syndecan-4 cytoplasmic domain is a dramatic conformational change near the C2 region that ablates an interaction site with the PDZ domain of syntenin. Wound healing studies further suggest that syndecan-4 phosphorylation might influence cell migration behavior. We conclude that the phosphorylation (Ser183) of syndecan-4 can play a critical role as a molecular switch to regulate its functions through conformational change.
Assuntos
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/química , Proteoglicanas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Células CHO , Movimento Celular , Cricetinae , Ativação Enzimática , Glicoproteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteína Quinase C-alfa/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/genética , Ratos , Eletricidade Estática , Especificidade por Substrato , Sindecana-4RESUMO
[This corrects the article DOI: 10.1371/journal.pone.0156296.].
RESUMO
Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.
Assuntos
Escherichia coli/genética , Proteínas Ligantes de Maltose/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Animais , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Humanos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Solubilidade , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
The human protein tyrosine kinase-6 (PTK6) polypeptide that is deduced from the cDNA sequence contains a Src homology (SH) 3 domain, SH2 domain, and catalytic domain of tyrosine kinase. We initiated biochemical and NMR characterization of PTK6 SH3 domain in order to correlate the structural role of the PTK6 using circular dichroism and heteronuclear NMR techniques. The circular dichroism data suggested that the secondary structural elements of the SH3 domain are mainly composed of beta-sheet conformations. It is most stable when the pH is neutral based on the pH titration data. In addition, a number of cross peaks at the low-field area of the proton chemical shift of the NMR spectra indicated that the PTK6 SH3 domain retains a unique and folded conformation at the neutral pH condition. For other pH conditions, the SH3 domain became unstable and aggregated during NMR measurements, indicating that the structural stability is very sensitive to pH environments. Both the NMR and circular dichroism data indicate that the PTK6 SH3 domain experiences a conformational instability, even in an aqueous solution.
Assuntos
Proteínas Tirosina Quinases/isolamento & purificação , Domínios de Homologia de src , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/enzimologia , Escherichia coli/genética , Glutationa Transferase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Human granulocyte colony-stimulating factor (hGCSF), a neutrophil-promoting cytokine, is an effective therapeutic agent for neutropenia patients who have undergone several cancer treatments. Efficient production of hGCSF using E. coli is challenging because the hormone tends to aggregate and forms inclusion bodies. This study examined the ability of seven different N-terminal fusion tags to increase expression of soluble hGCSF in E. coli. Four tag proteins, namely maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), increased the solubility of hGCSF under normal conditions. Lowering the expression temperature from 30°C to 18°C also increased the solubility of thioredoxin-tagged and glutathione S-transferase-tagged hGCSF. By contrast, hexahistidine-tagged hGCSF was insoluble at both temperatures. Simple conventional chromatographic methods were used to purify hGCSF from the overexpressed PDIb'a'-hGCSF and MBP-hGCSF proteins. In total, 11.3 mg or 10.2 mg of pure hGCSF were obtained from 500 mL cultures of E. coli expressing PDIb'a'-hGCSF or MBP-hGCSF, respectively. SDS-PAGE analysis and silver staining confirmed high purity of the isolated hGCSF proteins, and the endotoxin levels were less than 0.05 EU/µg of protein. Subsequently, the bioactivity of the purified hGCSF proteins similar to that of the commercially available hGCSF was confirmed using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50s of the cell proliferation dose-response curves for hGCSF proteins purified from MBP-hGCSF and PDIb'a'-hGCSF were 2.83±0.31 pM, and 3.38±0.41 pM, respectively. In summary, this study describes an efficient method for the soluble overexpression and purification of bioactive hGCSF in E. coli.
Assuntos
Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proliferação de Células , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/isolamento & purificação , HumanosRESUMO
Crotamine is a peptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus. Interestingly, crotamine demonstrates promising anticancer, antimicrobial, and antifungal activities. The crotamine peptide can also deliver plasmids into rapidly dividing cells, such as cancer and stem cells, and demonstrates potent analgesic effects. Efficiently producing crotamine in mammalian cells is difficult because it is both cell-permeable and cytotoxic. Prokaryotic expression of this peptide is also difficult to maintain because it does not fold properly in the cytoplasm, resulting in aggregation and in the formation of inclusion bodies. In our current study, we show for the first time that N-terminal fusion with three protein tags-N-utilization substance protein A (NusA), protein disulfide isomerase b'a' domain (PDIb'a'), and maltose-binding protein (MBP)-enables the soluble overexpression of crotamine in the cytoplasm of Escherichia coli. MBP-tagged crotamine was purified using Ni affinity, anion exchange, and MBP chromatography. The tag was cleaved using TEV protease, and the final product was pure on a silver-stained gels. In total, 0.9 mg pure crotamine was obtained from each liter of bacterial culture with endotoxin level approximately 0.15 EU/µg, which is low enough to use in biomedical applications. The identity and intramolecular disulfide bonds were confirmed using MALDI-TOF MS analysis. Purified crotamine inhibited the hKv1.3 channel (but not hKv1.5) in a dose-dependent manner with IC50 value of 67.2 ± 44.7 nM (n = 10), indicating the correct protein folding. The crotamine product fused with MBP at its N-terminus also inhibited the hKv1.3 channel, suggesting that the N-terminus is not involved in the channel binding of the toxin.
Assuntos
Venenos de Crotalídeos/análise , Canal de Potássio Kv1.3/antagonistas & inibidores , Proteínas Ligantes de Maltose/metabolismo , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/metabolismo , Escherichia coli , Concentração Inibidora 50 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, â¼37 mg, â¼12 mg, and â¼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.
Assuntos
Hormônio do Crescimento Humano/isolamento & purificação , Proteínas Ligantes de Maltose/metabolismo , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Escherichia coli/metabolismo , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/farmacologia , Humanos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Células Procarióticas/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Extracellular superoxide dismutase (EC-SOD) is the only enzyme that removes superoxide radical in the extracellular space. The reduction of EC-SOD is linked to many diseases, suggesting that the protein may have therapeutic value. EC-SOD is reported to be insoluble and to make inclusion bodies when overexpressed in the cytoplasm of Escherichia coli. The refolding process has the advantage of high yield, but has the disadvantage of frequent aggregation or misfolding during purification. For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and protein disulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli. MBP-tagged human EC-SOD (hEC-SOD) was purified by MBP affinity and anion exchange chromatography, and its identity was confirmed by MALDI-TOF MS analysis. The purified protein showed good enzyme activity in vitro; however, there was a difference in metal binding. When copper and zinc were incorporated into hEC-SOD before MBP tag cleavage, the enzymatic activity was higher than when the metal ions were bound to the purified protein after MBP tag cleavage. Therefore, the enzymatic activity of hEC-SOD is associated with metal incorporation and protein folding via disulfide bond.
Assuntos
Cobre/química , Dissulfetos/química , Escherichia coli/genética , Superóxido Dismutase/química , Zinco/química , Sequência de Aminoácidos , Cobre/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espaço Extracelular , Expressão Gênica , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição , Zinco/metabolismoRESUMO
Among the members of the fibroblast growth factor (FGF) family that affect the growth, differentiation, migration, and survival of many cell types, FGF2 is the most abundant in the central nervous system. Because of its wound healing effects, FGF2 has potential as a therapeutic agent. The protein is also added to the culture media to maintain stem cells. Expression and purification procedures for FGF2 that are highly efficient and low cost have been intensively investigated for the past two decades. Our current study focuses on the purification of FGF2 fused with b'a' domains of human protein disulfide isomerase to elevate overexpression, solubility, and stability with a simplified experimental procedure using only ion exchange chromatography, as well as on the confirmation of the biological activity of FGF2 on fibroblast Balb/c 3T3 cells and hippocampal neural cells.
Assuntos
Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Neurônios/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequência de Aminoácidos , Animais , Células Cultivadas , Escherichia coli/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Neurônios/citologia , Neurônios/metabolismo , Plasmídeos , Isomerases de Dissulfetos de Proteínas/metabolismo , Engenharia de Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/µg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.
Assuntos
Escherichia coli/genética , Expressão Gênica , Fator Inibidor de Leucemia/genética , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Ordem dos Genes , Humanos , Fator Inibidor de Leucemia/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Plasmídeos/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeAssuntos
Proteínas Arqueais/química , Thermoplasma/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Termodinâmica , Thermoplasma/metabolismoRESUMO
H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-ß-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.