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1.
Bioresour Technol ; 291: 121812, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31376668

RESUMO

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkLf) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkLf and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ±â€¯7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ±â€¯1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.


Assuntos
Proteínas de Membrana Transportadoras , Engenharia Metabólica , Escherichia coli , Oxigenases de Função Mista , Sinais Direcionadores de Proteínas
2.
J Med Life ; 12(2): 184-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406522

RESUMO

Hypoparathyroidism is a rare endocrine disease which is characterized by the deficiency of serum calcium levels. RhPTH is prescribed as a therapy for the management of refractory hypoparathyroidism. The aim of this study is to investigate 32 signal peptides of gram-negative bacterial origin and evaluate their potential for efficient secretion of recombinant human PTH (1-84)In E.coli to obtain higher expression of recombinant PTH in bacterial systems by using this fusion partner. SignalP and ProtParam servers were employed to predict the presence and location of signal peptide cleavage sites in protein sequence and computation of various physical and chemical parameters of protein respectively. Also, SOLpro server was applied for prediction of the protein solubility. Then ProtComp and SecretomeP online servers were employed to determine protein location. The evaluations showed that theoretically two signal peptides Lipopolysaccharide export system protein LptA (lptA) and Periplasmic pH-dependent serine endoprotease DegQ (degQ) are the most appropriate signal peptides examined. Due to the lack of post-translational modification in PTH, its periplasmic expression has preferences. Based on the results of this study, using bioinformatics and reliable servers signal peptides with appropriate secretory potential can be obtained which lead to the highest expression level.


Assuntos
Biologia Computacional/métodos , Escherichia coli/metabolismo , Hormônio Paratireóideo/metabolismo , Periplasma/metabolismo , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Simulação por Computador , Proteínas de Escherichia coli/metabolismo , Humanos , Solubilidade
3.
Nat Commun ; 10(1): 2586, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197146

RESUMO

Bacteria control gene expression in concert with their population density by a process called quorum sensing, which is modulated by bacterial chemical signals and environmental factors. In the human pathogen Streptococcus pyogenes, production of secreted virulence factor SpeB is controlled by a quorum-sensing pathway and environmental pH. The quorum-sensing pathway consists of a secreted leaderless peptide signal (SIP), and its cognate receptor RopB. Here, we report that the SIP quorum-sensing pathway has a pH-sensing mechanism operative through a pH-sensitive histidine switch located at the base of the SIP-binding pocket of RopB. Environmental acidification induces protonation of His144 and reorganization of hydrogen bonding networks in RopB, which facilitates SIP recognition. The convergence of two disparate signals in the SIP signaling pathway results in induction of SpeB production and increased bacterial virulence. Our findings provide a model for investigating analogous crosstalk in other microorganisms.


Assuntos
Proteínas de Bactérias/metabolismo , Exotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Percepção de Quorum/fisiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Animais , Modelos Animais de Doenças , Feminino , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Sinais Direcionadores de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Infecções Estreptocócicas/mortalidade , Streptococcus pyogenes/fisiologia , Virulência/fisiologia
4.
Comput Biol Chem ; 80: 225-233, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999249

RESUMO

BACKGROUND: The recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing. OBJECTIVE: The aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli. METHODS: Thirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package. RESULTS AND CONCLUSION: Computational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.


Assuntos
Simulação por Computador , Fator 7 de Crescimento de Fibroblastos/química , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/química , Sequência de Aminoácidos , Escherichia coli/química , Fator 7 de Crescimento de Fibroblastos/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , RNA Mensageiro/química , Proteínas Recombinantes/genética , Solubilidade , Termodinâmica
5.
Mol Biotechnol ; 61(6): 451-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997666

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
6.
J Plant Physiol ; 237: 12-20, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999073

RESUMO

Functions of domains or motifs, which are encoded by the transit peptide (TP) of the precursor of the small subunit of Rubisco (prSSU), have been investigated intensively in dicots. Functional characterization of the prSSU TP, however, is still understudied in maize. In this study, we found that the TP of maize prSSU1 did not function fully in chloroplast targeting in Arabidopsis or vice versa, indicating the divergent function of TPs in chloroplast targeting between maize and Arabidopsis. Through deletion or substitution assays, we found that the N-terminal region of maize or Arabidopsis prSSU1 was necessary and sufficient for importing specifically the fused-green fluorescent protein (GFP) into each corresponding chloroplast. Finally, we found that the first-five amino acids and MM motif in the N-terminal domain of the maize TP played an essential role in maize chloroplast targeting. Thus, our analyses demonstrate that the N-terminal domain of the prSSU1 TP is the key determinant in chloroplast targeting between maize and Arabidopsis. Our study highlights the unique properties of the maize prSSU1 TP in chloroplast targeting, thus helping to understand the role of N-terminal domain in chloroplast targeting across species. It will help to manipulate chloroplast transit peptides (cTPs) for crop bioengineering.


Assuntos
Arabidopsis/genética , Proteínas de Cloroplastos/genética , Sinais Direcionadores de Proteínas/genética , Ribulose-Bifosfato Carboxilase/genética , Zea mays/genética , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Zea mays/enzimologia , Zea mays/metabolismo
7.
Microb Cell Fact ; 18(1): 69, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971250

RESUMO

BACKGROUND: Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low. RESULTS: In this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL-1, which is the highest level that has been reported to date. CONCLUSIONS: This is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.


Assuntos
Bacillus subtilis/genética , Geobacillus stearothermophilus/enzimologia , Chaperonas Moleculares/genética , Sinais Direcionadores de Proteínas , alfa-Amilases/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Fermentação , Expressão Gênica , Geobacillus stearothermophilus/genética , Microbiologia Industrial , Chaperonas Moleculares/metabolismo , Mutação , Regiões Promotoras Genéticas , alfa-Amilases/metabolismo
8.
Microb Cell Fact ; 18(1): 72, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30995928

RESUMO

BACKGROUND: In terms of protein production, the internal environment of the host influences the activity of expression elements, thus affecting the expression level of the target protein. Native expression elements from a specific strain always function well in the original host. In the present study, to enhance the endoxylanase (XynA) production level in Corynebacterium glutamicum CGMCC1.15647 with its native expression elements, approaches to reduce host expression obstacles and to promote expression were evaluated. RESULTS: We identified the signal peptide of CspB2 in C. glutamicum CGMCC1.15647 by MALDI-TOF and applied it along with its promoter for the production of endoxylanase (XynA) in this strain. The native cspB2 promoter and cspB2 signal peptide are superior to the well-used cspB1 promoter and cspA signal peptide for XynA expression in C. glutamicum CGMCC1.15647, and expression in this strain is superior to the expression in C. glutamicum ATCC13032. The highest XynA secretion efficiency level in deep 24-well plates level (2492.88 U/mL) was achieved by disruption of the cell wall protein CspB2 and the protease ClpS, chromosomal integration of xynA and coexisting plasmid expression, which increased expression 11.43- and 1.35-fold compared to that of chromosomal expression and pXMJ19-xynA-mediated expression in the original strain, respectively. In fed-batch cultivation, the highest XynA accumulation (1.77 g/L) was achieved in the culture supernatant after 44 h of cultivation. CONCLUSION: Adaptation between the expression elements and the host is crucial for XynA production in C. glutamicum CGMCC1.15647. Strategies including host optimization, chromosomal integration, and coexistence of plasmids were useful for efficient protein production in C. glutamicum.


Assuntos
Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Engenharia Metabólica , Plasmídeos , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas
9.
J Microbiol Biotechnol ; 29(4): 625-632, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30954032

RESUMO

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding ß-agarase and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro- L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.


Assuntos
Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sefarose/metabolismo , Meios de Cultura , Dissacaridases/genética , Dissacarídeos/metabolismo , Enzimas/genética , Escherichia coli/genética , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Glicosídeo Hidrolases/genética , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo
10.
J Biotechnol ; 296: 22-31, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30878516

RESUMO

In previous studies of Lactococcus lactis, the levels of proteins secreted using heterologous signal peptides were observed to be lower than those obtained using the signal peptide from Usp45, the major secreted lactococcal protein. In this study, G1 (the native signal peptide of CGTase) and the signal peptide M5 (mutant of the G1 signal peptide) were introduced into L. lactis to investigate the effect of signal peptides on lactococcal protein secretion to improve secretion efficiency. The effectiveness of these signal peptides were compared to the Usp45 signal peptide. The highest secretion levels were obtained using the G1 signal peptide. Sequence analysis of signal peptide amino acids revealed that a basic N-terminal signal peptide is not absolutely required for efficient protein export in L. lactis. Moreover, the introduction of a helix-breaking residue in the H-region of the M5 signal peptide caused a reduction in the signal peptide hydrophobicity and decreased protein secretion. In addition, the optimization of cultivation conditions for recombinant G1-CGTase production via response surface methodology (RSM) showed that CGTase activity increased approximately 2.92-fold from 5.01 to 16.89 U/ml compared to the unoptimized conditions.


Assuntos
Proteínas de Bactérias/genética , Glucosiltransferases/efeitos dos fármacos , Lactococcus lactis/enzimologia , Sinais Direcionadores de Proteínas/genética , Proteínas de Bactérias/química , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Transporte Proteico/genética
11.
Rapid Commun Mass Spectrom ; 33(11): 1015-1023, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30884002

RESUMO

RATIONALE: Proteins undergo post-translational modifications and proteolytic processing that can affect their biological function. Processing often involves the loss of single residues. Cleavage of signal peptides from the N-terminus is commonly associated with translocation. Recent reports have suggested that other processing sites also exist. METHODS: The secreted proteins from S. aureus N315 were precipitated with trichloroacetic acid (TCA) and amidinated with S-methyl thioacetimidate (SMTA). Amidinated proteins were digested with trypsin and analyzed with a high-resolution orbitrap mass spectrometer. RESULTS: Sixteen examples of Staphylococcus aureus secretory proteins that lose an N-terminal signal peptide during their export were identified using this amidination approach. The N-termini of proteins with and without methionine were identified. Unanticipated protein cleavages due to sortase and an unknown protease were also uncovered. CONCLUSIONS: A simple N-terminal amidination based mass spectrometry approach is described that facilitates identification of the N-terminus of a mature protein and the discovery of unexpected processing sites.


Assuntos
Proteínas de Bactérias/química , Staphylococcus aureus/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Biocatálise , Butiratos/química , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Proteólise , Compostos de Sulfidrila/química , Ácido Tricloroacético/química , Tripsina/química
12.
Sheng Wu Gong Cheng Xue Bao ; 35(3): 425-434, 2019 Mar 25.
Artigo em Chinês | MEDLINE | ID: mdl-30912351

RESUMO

We constructed bicistronic expression system containing AH6 promoter, 5' UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃, respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4-11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.


Assuntos
Corynebacterium glutamicum , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Transporte Proteico
13.
Fish Shellfish Immunol ; 88: 352-358, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30851450

RESUMO

Antimicrobial peptides (AMP) are potential alternatives to conventional antibiotics with the prospect to treat infections caused by multidrug resistant bacteria. This is the report of the first arasin sequence from the mud crab, Scylla serrata, designated as Ss-arasin. The complete cDNA sequences of the open reading frame (ORF) is comprised of 198 bp encoding 65 amino acid with a predicted molecular weight of 7 kDa and a predicted isoelectric point of 10.68. The sequence of the N-terminal 24 amino acid residues is indicative of a signal sequence directing the newly synthesize protein toward the secretory pathway. The 41-residue mature peptide is composed of two domains, an N-terminal Gly/Arg-rich domain and a C-terminal cysteine-rich domain. Challenging the mud crab with lipopolysaccharide (LPS) increased expression of Ss-arasin mRNA in haemocytes, reaching the highest level at 6 h, before dropping to basal levels at 24 h. Recombinant rSs-arasin showed antimicrobial activity against three bacterial species Staphylococcus aureus (40 mM), Pseudomonas aeruginosa (40 mM) and Escherichia coli (40 mM) implying significant anti-bacterial action. In addition, recombinant rSs-arasin inhibited human cervical carcinoma (HeLa) and colon carcinoma (HT-29) cell growth. These initial findings are encouraging to further study the structure-activity relationships to optimize these biological functions for future drug development.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/efeitos dos fármacos , Braquiúros/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antineoplásicos/farmacologia , Clonagem Molecular , Descoberta de Drogas , Células HT29 , Células HeLa , Hemócitos/metabolismo , Humanos , Lipopolissacarídeos , Fases de Leitura Aberta , Sinais Direcionadores de Proteínas , RNA Mensageiro/metabolismo , Alinhamento de Sequência
14.
Appl Microbiol Biotechnol ; 103(8): 3341-3353, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30887174

RESUMO

Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Fosfatase Alcalina/genética , Afinidade de Anticorpos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Meios de Cultura/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
15.
BMC Biotechnol ; 19(1): 17, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894163

RESUMO

BACKGROUND: Parageobacillus thermoglucosidasius is a thermophilic and ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. The organism has been proposed to be a suitable organism for the production of bioethanol from lignocellulosic feedstocks. These feedstocks may be difficult to degrade, and a potential strategy to optimise this process is to engineer strains that secrete hydrolases that liberate increased amounts of sugars from those feedstocks. However, very little is known about protein transport in P. thermoglucosidasius and the limitations of that process, and as a first step we investigated whether there were bottlenecks in the secretion of a model protein. RESULTS: A secretory enzyme, xylanase (XynA1), was produced with and without its signal peptide. Cell cultures were fractionated into cytoplasm, membrane, cell wall, and extracellular milieu protein extracts, which were analysed using immunoblotting and enzyme activity assays. The main bottleneck identified was proteolytic degradation of XynA1 during or after its translocation. A combination of mass spectrometry and bioinformatics indicated the presence of several proteases that might be involved in this process. CONCLUSION: The creation of protease-deficient strains may be beneficial towards the development of P. thermoglucosidasius as a platform organism for industrial processes.


Assuntos
Geobacillus/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/administração & dosagem , Xilosidases/metabolismo , Células Cultivadas , Líquido Extracelular , Geobacillus/genética , Peptídeo Hidrolases/análise , Sinais Direcionadores de Proteínas , Proteólise , Proteoma/análise , Xilosidases/análise
16.
World J Microbiol Biotechnol ; 35(4): 54, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30900052

RESUMO

Filamentous fungi are important microorganisms used in industrial production of proteins and enzymes. Among these organisms, Trichoderma reesei, Aspergilli, and more recently Myceliophthora thermophile are the most widely used and promising ones which have powerful protein secretion capability. In recent years, there have been tremendous achievements in understanding the molecular mechanisms of the secretory pathways in filamentous fungi. The acquired pieces of knowledge can be harnessed to enhance protein production in filamentous fungi with assistance of state-of-the-art genetic engineering techniques.


Assuntos
Proteínas Fúngicas/biossíntese , Fungos/metabolismo , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Aspergillus/metabolismo , Códon , Proteínas Fúngicas/genética , Fungos/genética , Fungos/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Glicosilação , Peptídeos/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Saccharomycetales/metabolismo , Via Secretória/genética , Trichoderma/metabolismo
17.
Int J Mol Sci ; 20(5)2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818831

RESUMO

In the present study, we cloned, sequenced, and explored the structural and functional characteristics of the major histocompatibility complex (MHC)-DQA gene from mink (Neovison vison) for the first time. The full-length sequence of DQA gene was 1147-bp-long, contained a coding region of 768-bp, which was predicted to encoding 255 amino acid residues. The comparison between DQA from mink (Neovison vison) and other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of the mink DQA gene exhibited high similarity with the ferret (Mustela pulourius furo). Phylogenetic analysis revealed that mink (Neovison vison) DQA is grouped with that of ferret (Mustela pulourius furo). The cloned sequence contained a 23-amino acid NH2-terminal signal sequence with the signal peptide cutting site located in amino acids 23⁻24, and had three Asn-Xaa-Ser/Thr sequons. Three cysteine residues were also identified (Cys-85, Cys-121, and Cys-138). The 218 to 240 amino acids were predicted to be the transmembrane domains. The prediction of the secondary structure revealed three α-helixes and fourteen ß-sheets in Neovison vison DQA protein, while random coil was a major pattern. In this study, the whole CDS sequence of Neovison vison DQA gene was successfully cloned, which was valuable for exploring the function and antiviral molecular mechanisms underlying the molecule. The findings of the present study have laid the foundation for the disease resistance and breeding of mink.


Assuntos
Clonagem Molecular/métodos , Biologia Computacional/métodos , Cadeias alfa de HLA-DQ/genética , Vison/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Glicosilação , Cadeias alfa de HLA-DQ/química , Cadeias alfa de HLA-DQ/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Fosforilação , Filogenia , Domínios Proteicos , Sinais Direcionadores de Proteínas , Estrutura Secundária de Proteína
18.
Mol Biol Rep ; 46(2): 2243-2257, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30759297

RESUMO

MGP-40 is a mammary gland-specific glycoprotein which is expressed during involution and is an important marker for mammary gland apoptosis. It is an inactive chitinase-like protein belonging to Glycosyl Hydrolase family 18. The present study reports sequence characterization, tissue-specific expression analysis, production of recombinant MGP-40 and its mutant (A117D and L119E) in both E. coli and COS1 cells for their chitin-binding and chitinase activity analysis. The cDNA of buffalo MGP-40 was cloned and sequenced which corresponded to 1803 bp with an open reading frame of 1152 bp (361 aa), signal sequence of 63 bp (21 aa), 5' and 3' UTR of 144 bp and 507 bp, respectively. The 3' UTR analysis revealed potential sites for high level expression and stability during involution. The half-life of buffalo MGP-40 was found to be 11.7 h. MGP-40 was highly expressed in mammary gland followed by small intestine, spleen and mammary epithelial cells. The purified recombinant MGP-40 and its mutant expressed in E.coli were observed to bind chitin efficiently, however, no chitinase activity was observed. Further, chitinase activity was also not observed by expressing mutant recombinant MGP-40 in COS1 cells ruling out the possible role of post-translational modifications. Structure-based in-silico mutagenesis by FoldX algorithm showed a drastic decrease in overall fold stability which might be a possible reason for inability to recover its activity. Therefore, chitinase activity could not be restored in MGP-40 even after reverting back two critical residues in active site which may be due to detrimental effect of mutations on structural stability.


Assuntos
Búfalos/metabolismo , Proteína 1 Semelhante à Quitinase-3/metabolismo , Proteína 1 Semelhante à Quitinase-3/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Búfalos/genética , Búfalos/fisiologia , Células COS , Cercopithecus aethiops , Proteína 1 Semelhante à Quitinase-3/genética , Quitinases/genética , Quitinases/metabolismo , Clonagem Molecular/métodos , DNA Complementar/genética , Escherichia coli/genética , Feminino , Glicoproteínas/genética , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/fisiologia , Fases de Leitura Aberta , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética
19.
Methods Mol Biol ; 1960: 123-138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30798527

RESUMO

Outer membrane and secreted proteins in Gram-negative bacteria constitute a high percentage of virulence factors that are critical in disease initiation and progression. Despite their importance, it is often difficult to study these proteins due to challenges with expression and purification. Here we present a suite of vectors for the inducible expression of N-terminally 6His-tagged outer membrane, periplasmic, and secreted proteins in E. coli and show this system to be capable of producing milligram quantities of pure protein for downstream functional and structural analysis. This system can not only be used to purify recombinant virulence factors for structural and functional studies but can also be used to create gain-of-function E. coli for use in phenotypic screens, and examples of each are provided herein.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Periplasma/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia
20.
J Biol Chem ; 294(16): 6621-6634, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30792308

RESUMO

Nuclear localization of androgen receptor (AR) directs transcriptional regulation of a host of genes, referred to as genomic signaling. Additionally, nonnuclear or nongenomic activities of the AR have long been described, but understanding of these activities remains elusive. Here, we report that AR is imported into and localizes to mitochondria and has a novel role in regulating multiple mitochondrial processes. Employing complementary experimental approaches of AR knockdown in AR-expressing cells and ectopic AR expression in AR-deficient cells, we demonstrate an inverse relationship between AR expression and mitochondrial DNA (mtDNA) content and transcription factor A, mitochondrial (TFAM), a regulator of mtDNA content. We show that AR localizes to mitochondria in prostate tissues and cell lines and is imported into mitochondria in vitro We also found that AR contains a 36-amino-acid-long mitochondrial localization sequence (MLS) capable of targeting a passenger protein (GFP) to the mitochondria and that deletion of the MLS abolishes the import of AR into the mitochondria. Ectopic AR expression reduced the expression of oxidative phosphorylation (OXPHOS) subunits. Interestingly, AR also controlled translation of mtDNA-encoded genes by regulating expression of multiple nuclear DNA-encoded mitochondrial ribosomal proteins. Consistent with these observations, OXPHOS supercomplexes were destabilized, and OXPHOS enzymatic activities were reduced in AR-expressing cells and restored upon AR knockdown. Moreover, mitochondrial impairment induced AR expression and increased its translocation into mitochondria. We conclude that AR localizes to mitochondria, where it controls multiple mitochondrial functions and mitonuclear communication. Our studies also suggest that mitochondria are novel players in nongenomic activities of AR.


Assuntos
Mitocôndrias/metabolismo , Fosforilação Oxidativa , Sinais Direcionadores de Proteínas , Receptores Androgênicos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Células PC-3 , Transporte Proteico/genética , Receptores Androgênicos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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