-
European Journal of Cell Biology Aug 2018Signal peptides (SP) are short peptides located in the N-terminal of proteins, carrying information for protein secretion. They are ubiquitous to all prokaryotes and... (Review)
Review
Signal peptides (SP) are short peptides located in the N-terminal of proteins, carrying information for protein secretion. They are ubiquitous to all prokaryotes and eukaryotes. SPs have been of special interest in several scientific and industrial fields, including recombinant protein production, disease diagnosis, immunization, and laboratory techniques. Recently, the role of SPs in recombinant protein production has gained too much attention. Herein, several studies have been reviewed to elucidate the precise structure and function of SPs, particularly the optimized ones for recombinant protein production. However, some features of SPs still have remained obscure. In this review, some approaches concerning elucidation and optimization of SPs are discussed, and pragmatic conclusions and suggestions for future studies are also proposed. Moreover, a summary of secretory pathways, evolutionary changes, functions, applications, and different types of SPs is mentioned. At last, current limitations and prospects are discussed.
Topics: Animals; Humans; Protein Conformation; Protein Sorting Signals
PubMed: 29958716
DOI: 10.1016/j.ejcb.2018.06.003 -
Recent Patents on Biotechnology 2020Polyethylene terephthalate (PET) is the most widely produced polyester plastic in the world. PET is very difficult to catalyze or biological depolymerization due to the...
BACKGROUND
Polyethylene terephthalate (PET) is the most widely produced polyester plastic in the world. PET is very difficult to catalyze or biological depolymerization due to the limited access to ester bonds. Consequently, plastic will be stockpiled or flowed into the environment which is projected until hundreds of years. The most effective and environmental friendly plastic degradation method is biodegradation with microorganisms. Two specific enzyme for PET hydrolase, PETase and MHETase have been identified from Ideonella sakaiensis 201-F6. Recombinant genes are made to increase the effectiveness of enzymes in degrading PET. Previous studies of the PETase gene have been carried out, but to produce the final degradation PET product, the enzyme MHETase is needed. Thus, in this study the MHETase gene construction was carried out.
METHODS
The goal of this study is to construct MHETase gene in pUCIDT plasmid with native signal peptide from I. sakaensis 201-F6 and constitutive promoter J23106 was expressed in Escherichia coli BL21 (DE3) by heats shock. Expression analysis using SDS-PAGE and activity of enzyme is analyzed by spectrophotometry method and SEM.
RESULTS
MHETase gene protein was successfully constructed in pUCIDT +Amp plasmid with native signal peptide from Ideonella sakaensis 201-F6, T7 terminator and constitutive promoter J23106. PCR analysis showed that the gene successfully contained in the cells by band size (1813 bp) in electrophoresis gel. Analysis using Snap Gene, pairwise alignment using MEGA X, and NCBI was demonstrated that MHETase sequence the gene was in-frame in pUCIDT plasmid.
CONCLUSION
MHETase gene was successfully constructed in plasmids by in silico method. Synthetic plasmids transformed in E. coli BL21 (DE3) contain MHETase gene sequences which were in frame. Hence, the E. coli BL21 (DE3) cells have the potential to produce MHETase proteins for the plastic degradation testing process. We will patent the construct of MHETase gene using constitutive promoter and signal peptide from native which expressed in E. coli BL21 (DE3). This patent refers to a more applicable plastic degradation system with a whole cell without the need for purification and environmental conditioning of pure enzymes.
Topics: Amino Acid Sequence; Bacterial Proteins; Biodegradation, Environmental; Burkholderiales; Cloning, Molecular; Environmental Pollutants; Enzyme Assays; Escherichia coli; Gene Expression; Humans; Hydrolases; Industrial Microbiology; Kinetics; Patents as Topic; Plasmids; Polyethylene Terephthalates; Promoter Regions, Genetic; Protein Sorting Signals; Recombinant Proteins
PubMed: 32160855
DOI: 10.2174/1872208314666200311104541 -
PloS One 2015Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to...
Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.
Topics: Animals; Antibodies, Monoclonal; CHO Cells; Cricetulus; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Mass Spectrometry; Protein Sorting Signals
PubMed: 25706993
DOI: 10.1371/journal.pone.0116878 -
Science Advances Apr 2022Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, virulence cassette (PVC) was...
Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, virulence cassette (PVC) was proposed to inject toxins into eukaryotic cells by puncturing the cell membrane from outside. This makes it an ideal tool for protein delivery in biomedical research. However, how to manipulate this nanocomplex as a molecular syringe is still undetermined. Here, we identify that one group of N-terminal signal peptide (SP) sequences are crucial for the effector loading into the inner tube of PVC complex. By application of genetic operation, cryo-electron microscopy, in vitro translocation assays, and animal experiments, we show that, under the guidance of the SP, numerous prokaryotic and eukaryotic proteins can be loaded into PVC to exert their functions across cell membranes. We therefore might customize PVC as a potent protein delivery nanosyringe for biotherapy by selecting cargo proteins in a broad spectrum, regardless of their species, sizes, and charges.
Topics: Animals; Cryoelectron Microscopy; Photorhabdus; Polyvinyl Chloride; Protein Sorting Signals; Virulence
PubMed: 35486720
DOI: 10.1126/sciadv.abm2343 -
Nature Biotechnology Jul 2022Signal peptides (SPs) are short amino acid sequences that control protein secretion and translocation in all living organisms. SPs can be predicted from sequence data,...
Signal peptides (SPs) are short amino acid sequences that control protein secretion and translocation in all living organisms. SPs can be predicted from sequence data, but existing algorithms are unable to detect all known types of SPs. We introduce SignalP 6.0, a machine learning model that detects all five SP types and is applicable to metagenomic data.
Topics: Algorithms; Amino Acid Sequence; Language; Protein Sorting Signals; Proteins
PubMed: 34980915
DOI: 10.1038/s41587-021-01156-3 -
Nature Chemical Biology Sep 2023Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been...
Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.
Topics: Animals; Mice; Protein Sorting Signals; Protein Transport; Membrane Proteins; SEC Translocation Channels; Protein Biosynthesis
PubMed: 37169961
DOI: 10.1038/s41589-023-01326-1 -
Journal of Virology Oct 2023African swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007...
African swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007 through Russia, Eastern Europe, and more recently to Western European countries, and since 2018 emerged in China, from where it spread throughout Southeast Asia. Recently, outbreaks have appeared in the Caribbean, threatening the Americas. It is estimated that more than 900,000 animals have died directly or indirectly from ASFV since 2021 alone. One of the features of ASFV infection is hemoadsorption (HAD), which has been linked to virulence, although the molecular and pathological basis of this hypothesis remains largely unknown. In this study, we have analyzed and identified the key players responsible of HAD, contributing to the identification of new determinants of ASFV virulence, the understanding of ASFV pathogenesis, and the rational development of new vaccines.
Topics: Animals; African Swine Fever; African Swine Fever Virus; Glycosylation; Hemadsorption; Protein Sorting Signals; Swine; Virulence; Viral Proteins
PubMed: 37768082
DOI: 10.1128/jvi.01030-23 -
Protein Science : a Publication of the... Dec 2022Signal peptide (SP) plays an important role in membrane targeting for insertion of secretory and membrane proteins during translocation processes in prokaryotes and...
Signal peptide (SP) plays an important role in membrane targeting for insertion of secretory and membrane proteins during translocation processes in prokaryotes and eukaryotes. Beside the targeting functions, SP has also been found to affect the stability and folding of several proteins. Serum amyloid A (SAA) proteins are apolipoproteins responding to acute-phase inflammation. The fibrillization of SAA results in a protein misfolding disease named amyloid A (AA) amyloidosis. The main disease-associated isoform of human SAA, SAA1.1, is expressed as a precursor protein with an N-terminal signal peptide composed of 18 residues. The cleavage of the SP generates mature SAA1.1. To investigate whether the SP affects properties of SAA1.1, we systematically examined the structure, protein stability, and fibrillization propensity of pre-SAA1.1, which possesses the SP, and Ser-SAA1.1 without the SP but containing with an additional N-terminal serine residue. We found that the presence of the SP did not significantly affect the predominant helical structure but changed the tertiary conformation as evidenced by intrinsic fluorescence and exposed hydrophobic surfaces. Pre-SAA1.1 and Ser-SAA1.1 formed distinct oligomeric assemblies in which pre-SAA1.1 populated as tetramer and octamer, whereas Ser-SAA1.1 existed as a predominant hexamer. Pre-SAA1.1 was found significantly more stable than Ser-SAA1.1 upon thermal and chemical unfolding. Ser-SAA1.1, but not pre-SAA1.1, is capable of forming amyloid fibrils in protein misfolding study, indicating a protective role of the SP. Altogether, our results demonstrated a novel role of the SP in SAA folding and misfolding and provided a novel direction for therapeutic development of AA amyloidosis.
Topics: Humans; Serum Amyloid A Protein; Protein Sorting Signals; Amyloidosis; Amyloid
PubMed: 36309973
DOI: 10.1002/pro.4485 -
ACS Synthetic Biology Feb 2023The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology...
The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology and important industrial applications. Proteins are directed into the Sec pathway by a signal peptide at their N-terminus. Estimating the impact of physicochemical signal peptide features on protein secretion levels has not been achieved so far, partially due to the extreme sequence variability of signal peptides. To elucidate relevant features of the signal peptide sequence that influence secretion efficiency, an evaluation of ∼12,000 different designed signal peptides was performed using a novel miniaturized high-throughput assay. The results were used to train a machine learning model, and a post-hoc explanation of the model is provided. By describing each signal peptide with a selection of 156 physicochemical features, it is now possible to both quantify feature importance and predict the protein secretion levels directed by each signal peptide. Our analyses allow the detection and explanation of the relevant signal peptide features influencing the efficiency of protein secretion, generating a versatile tool for the de novo design and in silico evaluation of signal peptides.
Topics: Protein Sorting Signals; Bacillus subtilis; Protein Transport; Cell Membrane; Bacterial Proteins
PubMed: 36649479
DOI: 10.1021/acssynbio.2c00328 -
Methods in Enzymology 2017Signal peptidases are membrane proteases that play crucial roles in the protein transport pathway of bacteria. They cleave off the signal peptide from precursor proteins... (Review)
Review
Signal peptidases are membrane proteases that play crucial roles in the protein transport pathway of bacteria. They cleave off the signal peptide from precursor proteins that are membrane inserted by the SecYEG or Tat translocons. Signal peptide cleavage releases the translocated protein from the inner membrane allowing the protein to be exported to the periplasm, outer membrane, or secreted into the medium. Signal peptidases are very important proteins to study. They are unique serine proteases with a Ser-Lys dyad, catalyze cleavage at the membrane surface, and are promising potential antibacterial drug targets. This chapter will focus on the isolation of signal peptidases and the preprotein substrates, as well as describe a peptide library approach for characterizing the substrate specificity.
Topics: Amino Acid Sequence; Catalytic Domain; Membrane Proteins; Molecular Biology; Peptide Library; Periplasm; Protein Conformation; Protein Sorting Signals; Protein Transport; Sequence Homology, Amino Acid; Serine Endopeptidases; Serine Proteases; Staphylococcus aureus; Substrate Specificity
PubMed: 28065271
DOI: 10.1016/bs.mie.2016.09.025