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Chembiochem : a European Journal of... Jun 2022Nitrile hydratase (NHase) is an excellent biocatalyst for the synthesis of amide compounds and is composed of two heterologous subunits. However, the secretory...
Nitrile hydratase (NHase) is an excellent biocatalyst for the synthesis of amide compounds and is composed of two heterologous subunits. However, the secretory expression of NHase has been difficult to achieve because of its complex expression mechanism. In this work, a novel fluorescent probe Rho-IDA-CoII was synthesized by a one-pot method. Rho-IDA-CoII could specifically label His-tagged proteins in vitro, such as for staining in-gel, Western blot, and ELISA analysis. Furthermore, Rho-IDA-CoII combined with dot blots could quantitatively detect His-tagged proteins at between 1-10 pmol and perform high-throughput screening for the NHase signal peptide library. Recombinant Bacillus subtilis WB800/phoB-HBA with the extracellular expression of NHase was screened (ca. 6500 clones). After optimization of fermentation conditions, the NHase activity in the culture supernatant reached 17.34±0.16 U/mL. This is the first time that secretory NHase has been expressed in B. subtilis successfully.
Topics: Fluorescent Dyes; High-Throughput Screening Assays; Peptide Library; Protein Sorting Signals
PubMed: 35470527
DOI: 10.1002/cbic.202100523 -
BMC Genomics Jan 2022Proteases catalyze the hydrolysis of peptide bonds of proteins, thereby improving dietary protein digestibility, nutrient availability, as well as flavor and texture of...
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.
Topics: High-Throughput Screening Assays; Lactobacillus plantarum; Pediococcus; Pediococcus acidilactici; Peptide Hydrolases; Plasmids; Protein Sorting Signals
PubMed: 35021997
DOI: 10.1186/s12864-022-08292-3 -
Molecules (Basel, Switzerland) Oct 2022The COVID-19 pandemic is caused by SARS-CoV-2; the spike protein is a key structural protein that mediates infection of the host by SARS-CoV-2. In this study, we aimed...
The COVID-19 pandemic is caused by SARS-CoV-2; the spike protein is a key structural protein that mediates infection of the host by SARS-CoV-2. In this study, we aimed to evaluate the effects of signal peptide on the secretion and release of SARS-CoV-2 spike protein. Therefore, we constructed a signal peptide deletion mutant and three signal peptide site-directed mutants. The (H) region and (C) region in the signal peptide of L5F-S13I mutant have changed significantly, compared with wild type, L5F and S13I. We demonstrated the effects of signal peptide on the secretion and synthesis of RBD protein, finding that mutation of S13 to I13 on the signal peptide is more conducive to the secretion of RBD protein, which was mainly due to the shift of the signal peptide cleavage site in the mutant S13I. Here, we not only investigated the structure of the N-terminal signal peptide of the SARS-CoV-2 spike protein but also considered possible secretory pathways. We suggest that the development of drugs that target the signal peptide of the SARS-CoV-2 spike protein may have potential to treat COVID-19 in the future.
Topics: Humans; COVID-19; Pandemics; Protein Sorting Signals; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 36235223
DOI: 10.3390/molecules27196688 -
Antonie Van Leeuwenhoek Feb 1992
Review
Topics: Bacterial Proteins; Biological Transport; Endopeptidases; Escherichia; Protein Sorting Signals; Serratia marcescens
PubMed: 1580612
DOI: 10.1007/BF00580616 -
Journal of Molecular Microbiology and... Apr 2000The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the... (Review)
Review
The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery. It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid. The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes. The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide. A minimal Tat system requires a copy of tatA and a copy of tatC. The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer. The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes. The topology of evolution trees suggests a bacterial origin of the Tat system. In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway. However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances. Tat systems show a substrate-Tat component specificity and a species specificity. The pore size of the Tat channel is estimated as being between 5 and 9 nm. Operational models of the Tat system are proposed.
Topics: Arginine; Bacteria; Bacterial Proteins; Biological Evolution; Biological Transport, Active; Carrier Proteins; Escherichia coli Proteins; Gene Products, tat; Genes, Bacterial; Genes, tat; Membrane Transport Proteins; Models, Molecular; Operon; Protein Sorting Signals
PubMed: 10939242
DOI: No ID Found -
Applied and Environmental Microbiology May 2022Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low...
Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were , , , and , suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris. Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the gene product may facilitate the application of P. pastoris in industrial biocatalysis.
Topics: Pichia; Protein Sorting Signals; Recombinant Proteins; Saccharomycetales
PubMed: 35435711
DOI: 10.1128/aem.00296-22 -
BMC Bioinformatics Oct 2005The signal peptide plays an important role in protein targeting and protein translocation in both prokaryotic and eukaryotic cells. This transient, short peptide...
BACKGROUND
The signal peptide plays an important role in protein targeting and protein translocation in both prokaryotic and eukaryotic cells. This transient, short peptide sequence functions like a postal address on an envelope by targeting proteins for secretion or for transfer to specific organelles for further processing. Understanding how signal peptides function is crucial in predicting where proteins are translocated. To support this understanding, we present SPdb signal peptide database http://proline.bic.nus.edu.sg/spdb, a repository of experimentally determined and computationally predicted signal peptides.
RESULTS
SPdb integrates information from two sources (a) Swiss-Prot protein sequence database which is now part of UniProt and (b) EMBL nucleotide sequence database. The database update is semi-automated with human checking and verification of the data to ensure the correctness of the data stored. The latest release SPdb release 3.2 contains 18,146 entries of which 2,584 entries are experimentally verified signal sequences; the remaining 15,562 entries are either signal sequences that fail to meet our filtering criteria or entries that contain unverified signal sequences.
CONCLUSION
SPdb is a manually curated database constructed to support the understanding and analysis of signal peptides. SPdb tracks the major updates of the two underlying primary databases thereby ensuring that its information remains up-to-date.
Topics: Databases, Nucleic Acid; Internet; Peptides; Protein Sorting Signals; Protein Transport; Sequence Analysis, Protein; Systems Integration; User-Computer Interface; Vocabulary, Controlled
PubMed: 16221310
DOI: 10.1186/1471-2105-6-249 -
Biochemical and Biophysical Research... Sep 2022
Topics: Animals; Antibody Formation; CHO Cells; Codon; Cricetinae; Cricetulus; Protein Sorting Signals
PubMed: 35863090
DOI: 10.1016/j.bbrc.2022.06.072 -
Biochemical and Biophysical Research... Jan 2006TorD is the private chaperone of TorA, a periplasmic respiratory molybdoenzyme of Escherichia coli. In this study, it is demonstrated that TorD is required to maintain...
TorD is the private chaperone of TorA, a periplasmic respiratory molybdoenzyme of Escherichia coli. In this study, it is demonstrated that TorD is required to maintain the integrity of the twin-arginine signal sequence of the cytoplasmic TorA precursors. In the absence of TorD, 35 out of the 39 amino acid residues of the signal peptide were lost and the proteolysis of the N-terminal extremity of TorA precursors was not prevented by the molybdenum cofactor insertion. We thus propose that one of the main roles of TorD is to protect the TorA signal peptide to allow translocation of the enzyme by the TAT system.
Topics: Amino Acid Sequence; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Molecular Chaperones; Molecular Sequence Data; Oxidoreductases, N-Demethylating; Protein Sorting Signals
PubMed: 16337610
DOI: 10.1016/j.bbrc.2005.11.107 -
The EMBO Journal Mar 1987With few exceptions, the signal peptides from proteins inserted into, or translocated through, the membranes of gram-negative bacteria or the endoplasmic reticulum of...
With few exceptions, the signal peptides from proteins inserted into, or translocated through, the membranes of gram-negative bacteria or the endoplasmic reticulum of eukaryotes have no sequence homologies. Therefore these signal peptides have not been considered to contain information related to the different final localizations of the proteins. In this study, 43 signal peptide amino acid sequences from proteins with different final localizations in Escherichia coli have been subjected to a multivariate data analysis. Each amino acid residue was characterized by 20 physico-chemical properties, yielding a multivariate property profile for each peptide. The similarities/dissimilarities in the property profiles for the signal peptides from different classes were compared with each other by generating few-dimensional partial least squares (PLS) discriminant plots. With this approach, signal peptides from proteins localized to the periplasmic space (PS), the outer membrane (OM), and the extracellular surroundings (excreted proteins), were separated into distinct groups. Signal peptides from pili proteins were not separated from the OM signal peptides and only partly from the PS signal peptides, but were clearly different from the signal peptides of the excreted proteins. Signal peptides from inner membrane proteins were similar to those of the PS peptides. The size and the hydrophobicity of different peptide segments were responsible for the separation of the signal peptide classes. For example, the hydrophobicity of the N-terminal segment of the signal peptides increased with an increased distance from the cytoplasm of the final localization for the corresponding proteins. Thus, many signal peptides from proteins with different final localizations in E. coli have different discernible physico-chemical profiles.
Topics: Amino Acid Sequence; Analysis of Variance; Bacterial Proteins; Escherichia coli; Protein Sorting Signals
PubMed: 3556168
DOI: 10.1002/j.1460-2075.1987.tb04825.x