In Silico Investigation of Signal Peptide Sequences to Enhance Secretion of CD44 Nanobodies Expressed in Escherichia coli

2020 ◽  
Vol 21 ◽  
Author(s):  
Soudabeh Kavousipour ◽  
Shiva Mohammadi ◽  
Ebrahim Eftekhar ◽  
Mahdi Barazesh ◽  
Mohammad Hossein Morowvat
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Signal Peptide ◽  
In Silico ◽  
Extracellular Space ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Signal Peptide Sequence ◽  
E Coli ◽  
Peptide Database

Background: The selection of a suitable signal peptide that can direct recombinant proteins from the cytoplasm to the extracellular space is an important criterion affecting the production of recombinant proteins in Escherichia coli, a widely used host. Nanobodies are currently attracting the attention of scientists as antibody alternatives due to their specific properties and feasibility of production in E. coli. Objective: CD44 nanobodies constitute a potent therapeutic agent that can block CD44/HA interaction in cancer and inflammatory diseases. This molecule may also function as a drug against cancer cells and has been produced previously in E. coli without a signal peptide sequence. The goal of this project was to find a suitable signal peptide to direct CD44 nanobody extracellular secretion in E. coli that will potentially lead to optimization of experimental methods and facilitate downstream steps such as purification. Methods: We analyzed 40 E. coli derived signal peptides retrieved from the Signal Peptide database and selected the best candidate signal peptides according to relevant criteria including signal peptide probability, stability, and physicochemical features, which were evaluated using signalP software version 4.1 and the ProtParam tool, respectively. Results: In this in silico study, suitable candidate signal peptide(s) for CD44 nanobody secretory expression were identified. CSGA, TRBC, YTFQ, NIKA, and DGAL were selected as appropriate signal peptides with acceptable D-scores, and appropriate physicochemical and structural properties. Following further analysis, TRBC was selected as the best signal peptide to direct CD44 nanobody expression to the extracellular space of E. coli. Conclusion: The selected signal peptide, TRBC is the most suitable to promote high level secretory production of CD44 nanobodies in E. coli and potentially will be useful for scaling up CD44 nanobody production in experimental research as well as in other CD44 nanobody applications. However, experimental work is needed to confirm the data.

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.

scholarly journals EcDBS1R6: A new broad-spectrum cationic antibacterial peptide derived from a signal peptide sequence

2019 ◽  
Author(s):  
William F. Porto ◽  
Luz N. Irazazabal ◽  
Vincent Humblot ◽  
Evan F. Haney ◽  
Suzana M. Ribeiro ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Broad Spectrum ◽  
Antimicrobial Agents ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Peptide Sequence ◽  
World Health ◽  
Signal Peptides ◽  
Microscopy Imaging ◽  
Signal Peptide Sequence

ABSTRACTBacterial infections represent a major worldwide health problem, with an special highlight on Gram-negative bacteria, which were assigned by the World Health Organization (WHO) as the most critical priority for development of novel antimicrobial compounds. Antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the broad-spectrum activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes predicted through the Joker algorithm. Signal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker made this peptide capable of killing bacteria. Membrane damage of the bacterial cells was observed by measuring membrane integrity using fluorescent probes and through scanning electron microscopy imaging. Structural analysis revealed that the C-terminus was unable to fold into an α-helix, indicating that the EcDBS1R6 antibacterial activity core was located at the N-terminus, corresponding to the signal peptide portion of the parent peptide. Therefore, the strategy of transforming signal peptides into AMPs seems to be promising and could be used for producing novel antimicrobial agents.

scholarly journals Unexpected Diversity of Signal Peptides in Prokaryotes

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Samuel H. Payne ◽  
Stefano Bonissone ◽  
Si Wu ◽  
Roslyn N. Brown ◽  
Dmitry N. Ivankov ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Protein Localization ◽  
Cellular Protein ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Proteomics Data ◽  
Detailed Model ◽  
Signal Peptide Sequence ◽  
Peptide Motifs ◽  
Computational Analyses

ABSTRACT Signal peptides are a cornerstone mechanism for cellular protein localization, yet until now experimental determination of signal peptides has come from only a narrow taxonomic sampling. As a result, the dominant view is that Sec-cleaved signal peptides in prokaryotes are defined by a canonical AxA motif. Although other residues are permitted in the motif, alanine is by far the most common. Here we broadly examine proteomics data to reveal the signal peptide sequences for 32 bacterial and archaeal organisms from nine phyla and demonstrate that this alanine preference is not universal. Discoveries include fundamentally distinct signal peptide motifs from Alphaproteobacteria, Spirochaetes, Thermotogae and Euryarchaeota. In these novel motifs, alanine is no longer the dominant residue but has been replaced in a different way for each taxon. Surprisingly, divergent motifs correlate with a proteome-wide reduction in alanine. Computational analyses of ~1,500 genomes reveal numerous major evolutionary clades which have replaced the canonical signal peptide sequence with novel motifs. IMPORTANCE This article replaces a widely held general model with a more detailed model describing phylogenetically correlated variation in motifs for Sec secretion.

scholarly journals Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Garshasb Rigi ◽  
Amin Rostami ◽  
Habib Ghomi ◽  
Gholamreza Ahmadian ◽  
Vasiqe Sadat Mirbagheri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Hormone ◽  
Signal Peptide ◽  
Extracellular Space ◽  
Culture Medium ◽  
Active Form ◽  
Protein A ◽  
Human Growth ◽  
E Coli ◽  
Sds Page

Abstract Background Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. Conclusions Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

scholarly journals Revised Translation Start Site for secM Defines an Atypical Signal Peptide That Regulates Escherichia coli secA Expression

2000 ◽  
Vol 182 (19) ◽  
pp. 5592-5595 ◽  
Author(s):  
Shameema Sarker ◽  
Kenneth E. Rudd ◽  
Donald Oliver
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Peptide Sequence ◽  
Start Site ◽  
Signal Peptide Sequence ◽  
Amino Terminal ◽  
Upstream Regulator ◽  
Translation Start ◽  
Responsive Regulation ◽  
Translational Start

ABSTRACT The secretion-responsive regulation of Escherichia coli secA occurs by coupling its translation to the translation and secretion of an upstream regulator, secM (formerly geneX). We revise the translational start site for secM, defining a new signal peptide sequence with an extended amino-terminal region. Mutational studies indicate that certain atypical amino acyl residues within this extended region are critical for propersecA regulation.

scholarly journals Genome-wide high-throughput signal peptide screening via plasmid pUC256E improves protease secretion in Lactiplantibacillus plantarum and Pediococcus acidilactici

BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Binbin Chen ◽  
Bryan Zong Lin Loo ◽  
Ying Ying Cheng ◽  
Peng Song ◽  
Huan Fan ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Protein Digestibility ◽  
Fermented Food ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Pediococcus Acidilactici ◽  
Signal Peptide Sequence ◽  
Genome Wide ◽  
A Genome ◽  
Food And Feed

Abstract Background Proteases catalyze the hydrolysis of peptide bonds of proteins, thereby improving dietary protein digestibility, nutrient availability, as well as flavor and texture of fermented food and feed products. The lactobacilli Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) and Pediococcus acidilactici are widely used in food and feed fermentations due to their broad metabolic capabilities and safe use. However, extracellular protease activity in these two species is low. Here, we optimized protease expression and secretion in L. plantarum and P. acidilactici via a genetic engineering strategy. Results To this end, we first developed a versatile and stable plasmid, pUC256E, which can propagate in both L. plantarum and P. acidilactici. We then confirmed expression and secretion of protease PepG1 as a functional enzyme in both strains with the aid of the previously described L. plantarum-derived signal peptide LP_0373. To further increase secretion of PepG1, we carried out a genome-wide experimental screening of signal peptide functionality. A total of 155 predicted signal peptides originating from L. plantarum and 110 predicted signal peptides from P. acidilactici were expressed and screened for extracellular proteolytic activity in the two different strains, respectively. We identified 12 L. plantarum signal peptides and eight P. acidilactici signal peptides that resulted in improved yield of secreted PepG1. No significant correlation was found between signal peptide sequence properties and its performance with PepG1. Conclusion The vector developed here provides a powerful tool for rapid experimental screening of signal peptides in both L. plantarum and P. acidilactici. Moreover, the set of novel signal peptides identified was widely distributed across strains of the same species and even across some closely related species. This indicates their potential applicability also for the secretion of other proteins of interest in other L. plantarum or P. acidilactici host strains. Our findings demonstrate that screening a library of homologous signal peptides is an attractive strategy to identify the optimal signal peptide for the target protein, resulting in improved protein export.

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.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

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