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.

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 Precursors of select GPI-anchored proteins positively regulate GPI biosynthesis under the control of ERAD

2021 ◽  
Author(s):  
Yi-Shi Liu ◽  
Yicheng Wang ◽  
Xiaoman Zhou ◽  
LinPei Zhang ◽  
Ganglong Yang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Active Element ◽  
Signal Peptides ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Genome Wide ◽  
A Genome ◽  
Mechanistic Basis ◽  
Gpi Transamidase ◽  
Regulatory Connection

Abstract We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is regulated by endoplasmic reticulum associated degradation (ERAD); however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR–Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 together upregulate GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, remaining the uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 32 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55 and CD48 enhance GPI biosynthesis. ARV1 is essential for the GPI upregulation by CD55 precursor. Our data demonstrate an ARV1-dependent regulatory connection between GPI biosynthesis and precursors of select GPI-anchored proteins that are under the control of ERAD.

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.

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.

Cassette mutagenic analysis of the yeast invertase signal peptide: effects on protein translocation

1989 ◽  
Vol 9 (8) ◽  
pp. 3400-3410
Author(s):  
J K Ngsee ◽  
W Hansen ◽  
P Walter ◽  
M Smith
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Plasmid Vector ◽  
Peptide Sequence ◽  
Secretory Process ◽  
Hydrophobic Core ◽  
Class Iii ◽  
Yeast Plasmid ◽  
Signal Peptide Sequence ◽  
Transcription Terminator

The coding sequence of the SUC2 locus was placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere-based yeast plasmid vector from which invertase is expressed in a Suc- strain of Saccharomyces cerevisiae. Mutants in the signal peptide sequence were produced by replacing this region of the gene with synthetic oligonucleotide cassettes containing mixtures of nucleotides at several positions. The mutants could be divided into three classes on the basis of the ability to secrete invertase. Class I mutants produced secreted invertase but in reduced amount. The class II mutant, 4-55B, also exhibited reduced a level of invertase, but a significant fraction of the enzyme was intracellular. Class III mutants were partially defective in translocation from the cytoplasm to the endoplasmic reticulum and produced enzymatically active, unglycosylated preinvertase in the cytoplasm. Class III mutant preinvertases were also defective in translocation across canine pancreas microsomes. These results suggested that the reduced level of invertase resulted from proteolytic degradation of inefficiently transported intermediates. Comparison of the sequences of the mutant signal peptides indicated that amino acids at the extreme amino terminus and adjacent to the cleavage site play a crucial role in the secretory process when combined with a mutation within the hydrophobic core.

scholarly journals Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Doreen A. Wüstenhagen ◽  
Phil Lukas ◽  
Christian Müller ◽  
Simone A. Aubele ◽  
Jan-Peter Hildebrandt ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Peptide Sequence ◽  
Bacterial Cells ◽  
Recombinant Hirudin ◽  
Signal Peptide Sequence ◽  
Cell Lysates ◽  
Blood Sucking ◽  
A Cell ◽  
Synthesis Approach ◽  
Cell Free Protein Synthesis

AbstractSynthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells.

Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase

2019 ◽  
Vol 291 ◽  
pp. 121812 ◽  
Author(s):  
Hee-Wang Yoo ◽  
Joonwon Kim ◽  
Mahesh D. Patil ◽  
Beom Gi Park ◽  
Sung-yeon Joo ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Signal Peptide ◽  
Acid Methyl Ester ◽  
Peptide Sequence ◽  
Dodecanoic Acid ◽  
Signal Peptide Sequence ◽  
Acid Methyl

scholarly journals A New IL6 Isoform in Chinese Soft-Shelled Turtle (Pelodiscus sinesis) Discovered: Its Regulation during Cold Stress and Infection

Biology ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 111
Author(s):  
Zuobing Zhang ◽  
Miao Tian ◽  
Ruxin Song ◽  
Xiao Xing ◽  
Yong Fan ◽  
...  
Keyword(s):  
Cold Stress ◽  
Signal Peptide ◽  
Genomic Sequence ◽  
Adaptive Immune Response ◽  
Environmental Water ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Gene Characterization ◽  
Peptide Gene ◽  
Response To Temperature

The Chinese soft-shelled turtle (Pelodiscus sinesis) is a widely cultured commercial species in East and Southeast Asian countries. The turtles frequently suffer from acute cold stress during farming in China. Stress-induced factor such as Interleukin-6 (IL6) is a multifunctional molecule that plays important roles in innate and adaptive immune response. In the present study, we found that the turtle possessed two IL6 transcripts, where one IL6 transcript contained a signal peptide sequence (psIL6), while the other IL6 transcript (psIL6ns) possessed no such signal peptide gene. To test any differential expression of the two isoforms during temperature and microbial stress, turtles were adapted to optimal environmental water temperature (25 °C), stressed by acute cooling for 24 h, followed with the challenge of Aeromonas hydrophila (1.8 × 108 CFU) or Staphylococcus aureus (5.8 × 108 CFU). Gene characterization revealed that psIL6ns, a splicer without codons encoding a signal peptide and identical to the one predicted from genomic sequence, and psIL6, a splicer with codons encoding a signal peptide, were both present. Inducible expression was documented in primary spleen cells stimulated with ConA and poly I: C. The splenic and intestinal expression of psIL6ns and psIL6 was increased in response to temperature stress and bacterial infection.

scholarly journals Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from Streptococcus uberis

2002 ◽  
Vol 184 (1) ◽  
pp. 119-125 ◽  
Author(s):  
Philip N. Ward ◽  
James A. Leigh
Keyword(s):  
Plasminogen Activator ◽  
Signal Peptide ◽  
Open Reading Frame ◽  
Peptide Sequence ◽  
Expression Cloning ◽  
Signal Peptide Sequence ◽  
Putative Gene ◽  
Reading Frame ◽  
Streptococcus Uberis ◽  
Broad Specificity

ABSTRACT A bovine plasminogen activator of atypical molecular mass (∼45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072–1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pauA open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the pauB gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.

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