scholarly journals In silico analysis of signal peptides for secretory production of a-amylase in Bacillus subtilis

2019 ◽  
pp. 113-124
Author(s):  
Marzieh Asadi ◽  
Morgaza Taheri-Anganeh ◽  
Zeinab Jamali ◽  
Seyyed Hossein Khatami ◽  
Cambyz Irajie ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
In Silico ◽  
In Silico Analysis ◽  
Essential Elements ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Experimental Production ◽  
Secretory Production ◽  
Peptide Secretion

α-Amylases are important commercial enzymes and have a broad application in industrial processes and medicine. Gram-positive bacteria such as Bacillus subtilis are possible host organisms for α-amylases secretory production. Secretion of α-amylases to the culture medium versus intracellular production has several advantages such as prevention of inclusion bodies accumulation, higher product stability and solubility. Signal peptides are considered as one of the most essential elements for successful secretory synthesis of the recombinant proteins. Therefore, by the selection of an efficient signal peptide, secretion of the recombinant protein can be enhanced. The goal of this investigation was the in silico evaluation of several peptides to find the most suitable leader peptides for secretory production of α-amylase in B. subtilis. In present work, 30 signal peptides were selected, and numerous online servers such as SignalP, ProtParam, SOLpro, PRED-TAT and ProtComp was used for investigation of suitable signal peptides. According to in silico predictions all other signal peptides connected to α-amylase were stable and soluble except PPBD_BACSU. PPBD_BACSU because of having D-score below cut-off could not be recognized as a suitable signal peptide for α-amylase. Computational analysis identified QOX2_BACSU may direct protein into transmembrane location and was ignored. All 28 remained were predicted as secretory signal peptides which can excrete protein out of the bacteria. The signal peptides recommended by the present study are valuable for rational designing of secretory soluble α-amylase. Although, such information can be useful for future experimental production of these mentioned secretory proteins.

In silico Analysis of Different Signal Peptides for Secretory Production of Arginine Deiminase in Escherichia coli

2019 ◽  
Vol 13 (3) ◽  
pp. 217-227 ◽  
Author(s):  
Mahboubeh Zarei ◽  
Navid Nezafat ◽  
Mohammad Hossein Morowvat ◽  
Mohsen Ektefaie ◽  
Younes Ghasemi
Keyword(s):  
Physicochemical Properties ◽  
Signal Peptide ◽  
In Silico ◽  
In Silico Analysis ◽  
Cost Effective ◽  
Arginine Deiminase ◽  
Signal Peptides ◽  
E Coli ◽  
Cost Effective Alternative ◽  
Secretory Production

Background: Secretory production of recombinant protein in bacterial hosts fulfills several advantages. Selecting an appropriate secretory signal peptide is a critical step in secretory production of different protein. Several patents report the usage of signal peptides for secretory production of recombinant proteins in E. coli. In silico identification of suitable signal peptides is a reliable and cost-effective alternative to experimental approaches. Objective: This study was aimed to predict best signal peptides for the secretory production of recombinant arginine deiminase in E. coli. Methods: In this study, 30 different signal peptide sequences were retrieved from database. The signal peptide probability, location of cleavage sites, and n, h and c regions were predicted by SignalP 4.1 and Phobius servers. After purging the 30 predicted secretory signal peptides, TorT, bla, NrfA, TolB, PapC, PldA, Lpp were removed. Several physicochemical properties of the remaining potential SPs were determined by ProtParam, PROSO II, and SOLpro servers for theoretically selecting the best candidates. Results and Conclusion: Based on physicochemical properties, the signal peptides of OmpC, OmpF, and DsbA were identified respectively as the promising candidates for efficient secretory production of arginine deiminase in E. coli. Although the computational approach has established itself as a basis of modern biotechnology, the experimental study is necessary to validate its results. The criteria used in this study could be applied to other targets for recombination processes.

In Silico Evaluation of Various Signal Peptides to Improve Secretion of Humulin Protein in E. coli Host

2020 ◽  
Vol 17 ◽  
Author(s):  
Soudabe Kavousipour ◽  
Mahadi Barazesh ◽  
Shiva Mohammadi ◽  
Meghdad Abdollahpour- Alitappeh ◽  
Shirzad Fallahi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
In Silico ◽  
Signal Sequence ◽  
Expression Vectors ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Genetic Characteristics ◽  
E Coli

Background:: Escherichia coli host has been the workhorse for the production of heterologous proteins due to simplicity of use, low cost, availability of various expression vectors, and widespread knowledge on its genetic characteristics, but without a suitable signal sequence, this host cannot be used for production secretory proteins. Humulin is a form of insulin used to treat hyperglycemia caused by types 1 and 2 diabetes. To improve expression and make a straightforward production of Humulin protein, we chose a series of signal peptides. Objective:: aim our study to predict the most excellent signal peptides to express secretory Humulin in E. coli organisms. Method:: Therefore, to forecast the most excellent signal peptides for expression of Humulin in Escherichia coli, 47 signal sequences from bacteria organisms were elected and the most imperative elements of them were studied. Hence, signal peptide probability along with physicochemical features was evaluated by signal 4.1, and Portparam, PROSO II servers respectively. Later, the in-silico cloning in a known pET28a plasmid system also estimated the possibility of best signal peptide+ Humulin expression in E.Coli. Results:: The outcomes demonstrated among 47 signal peptides only 2 signal peptides can be suggested as suitable signal peptides. Conclusion:: Ultimately protein yebF precursor (YEBF_ECOLI) and protein yebF precursor (YEBF_YERP3) were suggested severally; as the most excellent signal peptides to express Humulin (With D scores 0.812 and 0.623 respectively). Although verification of these results want experimental analysis.

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli

2019 ◽  
Vol 80 ◽  
pp. 225-233 ◽  
Author(s):  
Mansoureh Shahbazi Dastjerdeh ◽  
Mahya Marashiyan ◽  
Mohammadtaghi Borjian Boroujeni ◽  
Majid Golkar ◽  
Mohammad Ali Shokrgozar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
In Silico ◽  
Keratinocyte Growth Factor ◽  
In Silico Analysis ◽  
Signal Peptides ◽  
Human Keratinocyte ◽  
Secretory Production ◽  
Silico Analysis

In Silico Evaluation of Different Signal Peptides for the Secretory Production of Human Growth Hormone in E. coli

2015 ◽  
Vol 21 (3) ◽  
pp. 261-268 ◽  
Author(s):  
Mozhdeh Zamani ◽  
Navid Nezafat ◽  
Manica Negahdaripour ◽  
Fatemeh Dabbagh ◽  
Younes Ghasemi
Keyword(s):  
Growth Hormone ◽  
Human Growth Hormone ◽  
In Silico ◽  
Human Growth ◽  
Signal Peptides ◽  
E Coli ◽  
Secretory Production

scholarly journals Identification of Novel Hypothalamic MicroRNAs as Promising Therapeutics for SARS-CoV-2 by Regulating ACE2 and TMPRSS2 Expression: An In Silico Analysis

2020 ◽  
Vol 10 (10) ◽  
pp. 666
Author(s):  
Debasmita Mukhopadhyay ◽  
Bashair M. Mussa
Keyword(s):  
In Silico ◽  
Binding Capacity ◽  
Therapeutic Targets ◽  
In Silico Analysis ◽  
Neurological Impairment ◽  
Essential Elements ◽  
Cell Entry ◽  
Angiotensin Converting Enzyme 2 ◽  
Transmembrane Serine Protease ◽  
Silico Analysis

Background: Neuroinvasion of severe acute respiratory syndrome coronavirus (SARS-CoV) is well documented and, given the similarities between this virus and SARS-CoV-2, it seems that the neurological impairment that is associated with coronavirus disease 2019 (COVID-19) is due to SARS-CoV-2 neuroinvasion. Hypothalamic circuits are exposed to the entry of the virus via the olfactory bulb and interact centrally with crucial respiratory nuclei. Hypothalamic microRNAs are considered as potential biomarkers and modulators for various diseases and future therapeutic targets. The present study aims to investigate the microRNAs that regulate the expression of hypothalamic angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential elements for SARS-CoV-2 cell entry. Methods: To determine potential hypothalamic miRNAs that can directly bind to ACE2 and TMPRSS2, multiple target bioinformatics prediction algorithms were used, including miRBase, Target scan, and miRWalk2.029. Results: Our in silico analysis has revealed that, although there are over 5000 hypothalamic miRNAs, around 31 miRNAs and 29 miRNAs have shown binding sites and strong binding capacity against ACE2 and TMPRSS2, respectively. Conclusion: These novel potential hypothalamic miRNAs can be used to identify new therapeutic targets to treat neurological symptoms in COVID-19 patients via regulation of ACE2 and TMPRSS2 expression.

scholarly journals Two Distinct Modes of Lysis Regulation in Campylobacter Fletchervirus and Firehammervirus Phages

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1247
Author(s):  
Athina Zampara ◽  
Stephen J. Ahern ◽  
Yves Briers ◽  
Lone Brøndsted ◽  
Martine Camilla Holst Sørensen
Keyword(s):  
Signal Peptide ◽  
In Silico ◽  
Protein Domains ◽  
In Silico Analysis ◽  
Cell Lysis ◽  
E Coli ◽  
Sec Pathway ◽  
Gene Functions ◽  
Lytic Genes ◽  
Silico Analysis

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.

scholarly journals RECONSTITUTION OF DIFFERENT SIGNAL PEPTIDES FOR ENHANCED THERMOSTABLE ALPHA AMYLASE SECRETION IN Bacillus subtilis

2018 ◽  
Vol 56 (1) ◽  
pp. 7 ◽  
Author(s):  
Nguyen Thị Da ◽  
Tran Dinh Man ◽  
Nguyen Kim Thoa
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Bacillus Cereus ◽  
Bacillus Licheniformis ◽  
Signal Peptide ◽  
Amylase Activity ◽  
Alpha Amylase ◽  
Amylase Secretion ◽  
Signal Peptides ◽  
Extracellular Amylase

Three signal peptides of alpha amylase genes of three isolated strains that were Bacillus licheniformis DA23, Bacillus subtilis D5-2, Bacillus cereus CN1-5 were successfully sequenced. Three predicted “Sec – type” signal peptides have a length varying from 27 (CN1-5) to 33 residues (D5-2).  The secretion of alpha amylase of the recombination B. subtilis 168MPgrac strain (pHV33–PgracAmy3BT2) with 71.4U/ml was larger than that of 168MPamy with 53.2U/ml. Base on analyzed rerults of PAGE and zymogram about molecular weight, alpha amylases in both strains were the same size, nearly 58kDa. The extracellular amylase activity of signal peptide SsubtilisD5.2 in 168M was highest with 76.4±4.3 U/ml in four signal peptide targets.  

scholarly journals Engineering of The Bacillus Subtilis PhoD Signal Peptide To Direct High-Level, Tat-Specific Export of A Single-Chain Antibody Fragment To The Periplasm in Eschericha Coli

2020 ◽  
Author(s):  
Chillel Jawara ◽  
Kirsty L Richards ◽  
Amber R Peswani ◽  
Kelly L Walker ◽  
Lara Nascimento ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Antibody Fragment ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Tat Pathway ◽  
Single Chain Antibody Fragment

Abstract Background: Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results: We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions: The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

scholarly journals Efficacy of signal peptide predictors in identifying signal peptides in the experimental secretome of Picrophilous torridus, a thermoacidophilic archaeon

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255826
Author(s):  
Neelja Singhal ◽  
Anjali Garg ◽  
Nirpendra Singh ◽  
Pallavi Gulati ◽  
Manish Kumar ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Signal Peptide ◽  
Carbon Metabolism ◽  
Metabolic Pathways ◽  
Protein Translocation ◽  
Mass Spectrometric ◽  
Secreted Proteins ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Microbial Adaptation

Secretory proteins are important for microbial adaptation and survival in a particular environment. Till date, experimental secretomes have been reported for a few archaea. In this study, we have identified the experimental secretome of Picrophilous torridus and evaluated the efficacy of various signal peptide predictors (SPPs) in identifying signal peptides (SPs) in its experimental secretome. Liquid chromatography mass spectrometric (LC MS) analysis was performed for three independent P. torridus secretome samples and only those proteins which were common in the three experiments were selected for further analysis. Thus, 30 proteins were finally included in this study. Of these, 10 proteins were identified as hypothetical/uncharacterized proteins. Gene Ontology, KEGG and STRING analyses revealed that majority of the sercreted proteins and/or their interacting partners were involved in different metabolic pathways. Also, a few proteins like malate dehydrogenase (Q6L0C3) were multi-functional involved in different metabolic pathways like carbon metabolism, microbial metabolism in diverse environments, biosynthesis of antibiotics, etc. Multi-functionality of the secreted proteins reflects an important aspect of thermoacidophilic adaptation of P. torridus which has the smallest genome (1.5 Mbp) among nonparasitic aerobic microbes. SPPs like, PRED-SIGNAL, SignalP 5.0, PRED-TAT and LipoP 1.0 identified SPs in only a few secreted proteins. This suggests that either these SPPs were insufficient, or N-terminal SPs were absent in majority of the secreted proteins, or there might be alternative mechanisms of protein translocation in P. torridus.

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