-
ACS Synthetic Biology Oct 2022DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for...
DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelB and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelB, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency.
Topics: Protein Sorting Signals; Signal Recognition Particle; Designed Ankyrin Repeat Proteins; Peptide Library; Hydrophobic and Hydrophilic Interactions; Bacteriophages; Thioredoxins; Codon; Valine
PubMed: 36178799
DOI: 10.1021/acssynbio.2c00260 -
Biotechnology Letters Jun 2018α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to...
OBJECTIVE
α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to construct recombinant Aspergillus niger strains with high α-galactosidase production.
RESULTS
Two recombinant A. niger strains were constructed: AB and AGB. The recombinant AB strain contained the α-galactosidase aglB gene from A. niger with its native AglB signal peptide regulated by the glucoamylase promoter. In the AGB recombinant strain, the AglB signal peptide was replaced with the glucoamylase (GlaA) signal peptide. The extracellular maximum α-galactosidase activity of the AGB strain was 215.7 U/ml and that of the AB strain was 9.8 U/mL. The optimal conditions for α-galactosidase were pH 3.5 and 35 °C.
CONCLUSIONS
The GlaA signal peptide substantially increased the yield of secreted α-galactosidase in A. niger. This recombinant strain holds great potential for industrial applications.
Topics: Aspergillus niger; Cloning, Molecular; Enzyme Stability; Escherichia coli; Fungal Proteins; Protein Sorting Signals; Recombinant Proteins; alpha-Galactosidase
PubMed: 29605944
DOI: 10.1007/s10529-018-2540-5 -
Journal of Virology Jul 2021Pestiviruses are members of the family , a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope...
Pestiviruses are members of the family , a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, E, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2. The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to E and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.
Topics: Amino Acid Sequence; Animals; Cattle; Cell Line; Cell Membrane; Cricetinae; Diarrhea Viruses, Bovine Viral; Endoplasmic Reticulum; Membrane Glycoproteins; Protein Conformation; Protein Sorting Signals; Rabbits; Viral Envelope Proteins
PubMed: 34011544
DOI: 10.1128/JVI.00521-21 -
Applied Microbiology and Biotechnology Jun 2022Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable...
Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain. KEY POINTS: • Automated workflows for strain construction and screening of protein secretion • Comparison of spacer, signal peptide, and host combinations for cutinase secretion • Signal peptide screening for secretion by C. glutamicum using the split GFP assay.
Topics: Automation, Laboratory; Bacillus subtilis; Corynebacterium glutamicum; Fusarium; Protein Sorting Signals; Protein Transport
PubMed: 35759036
DOI: 10.1007/s00253-022-12017-7 -
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 -
International Journal of Molecular... Dec 2021Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades... (Review)
Review
Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease.
Topics: Animals; Cytosol; Endoplasmic Reticulum; Humans; Protein Sorting Signals; Protein Transport; Proteins; Signal Recognition Particle
PubMed: 35008565
DOI: 10.3390/ijms23010143 -
Journal of Chromatography. B,... Nov 2017Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry...
Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry platforms and reverse phase chromatography. Detection and quantification of these variants by three LC/MS platforms were assessed. Quantification was also performed by mass spectrometric analysis of the subunits of the antibody generated by reduction and IdeS proteolysis. Peptide mapping with LC/MS/MS detection was used to quantify and confirm the identities of signal peptide sequence variants. Although quantification of the signal peptide variants thru mass spectrometry approaches is system dependent, our data revealed the results are close to the values determined by chromatographic separation with UV detection. Each of the methods have proven effective in demonstrating the consistency of signal peptide variants levels across the manufacture history of the antibody.
Topics: Animals; Antibodies, Monoclonal; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Immunoglobulin G; Molecular Weight; Peptide Mapping; Protein Sorting Signals; Tandem Mass Spectrometry
PubMed: 29078145
DOI: 10.1016/j.jchromb.2017.08.046 -
ACS Synthetic Biology Jun 2021Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of...
Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast . Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: UPO from and UPO from . To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from . In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide system, we made all plasmids available through Addgene.
Topics: Feasibility Studies; Fungal Proteins; High-Throughput Screening Assays; Microorganisms, Genetically-Modified; Mixed Function Oxygenases; Pichia; Plasmids; Promoter Regions, Genetic; Protein Engineering; Protein Sorting Signals; Recombinant Proteins; Saccharomycetales; Sordariales
PubMed: 34075757
DOI: 10.1021/acssynbio.0c00641 -
Methods in Molecular Biology (Clifton,... 2022Mammalian galectins have no signal peptide, and it is not known what would happen if a galectin is directed to take the classical export route. The corresponding...
Mammalian galectins have no signal peptide, and it is not known what would happen if a galectin is directed to take the classical export route. The corresponding engineering of galectin-specific cDNA will answer questions on the fate of a signal peptide-bearing protein variant after its entry into the endoplasmic reticulum (ER). Affinity chromatography and mass-spectrometric analysis of occupancy of potential N-glycosylation sites for the galectin, binding and functional assays with cells as well as subcellular fractionation by density gradient ultracentrifugation and immunocytochemical colocalization with ER/Golgi markers report on aspects of the consequences of letting a galectin enter new territory. Applying these methods will help to clarify why galectins are leaderless and thus produced by free ribosomes.
Topics: Animals; Endoplasmic Reticulum; Galectins; Glycosylation; Golgi Apparatus; Humans; Mammals; Protein Sorting Signals
PubMed: 35320531
DOI: 10.1007/978-1-0716-2055-7_15 -
ELife Jun 2021ER proteins of widely differing abundance are retrieved from the Golgi by the KDEL-receptor. Abundant ER proteins tend to have KDEL rather than HDEL signals, whereas...
ER proteins of widely differing abundance are retrieved from the Golgi by the KDEL-receptor. Abundant ER proteins tend to have KDEL rather than HDEL signals, whereas ADEL and DDEL are not used in most organisms. Here, we explore the mechanism of selective retrieval signal capture by the KDEL-receptor and how HDEL binds with 10-fold higher affinity than KDEL. Our results show the carboxyl-terminus of the retrieval signal moves along a ladder of arginine residues as it enters the binding pocket of the receptor. Gatekeeper residues D50 and E117 at the entrance of this pocket exclude ADEL and DDEL sequences. D50N/E117Q mutation of human KDEL-receptors changes the selectivity to ADEL and DDEL. However, further analysis of HDEL, KDEL, and RDEL-bound receptor structures shows that affinity differences are explained by interactions between the variable -4 H/K/R position of the signal and W120, rather than D50 or E117. Together, these findings explain KDEL-receptor selectivity, and how signal variants increase dynamic range to support efficient ER retrieval of low and high abundance proteins.
Topics: Endoplasmic Reticulum; Golgi Apparatus; Humans; Mutation; Protein Sorting Signals; Protein Transport; Receptors, Peptide
PubMed: 34137369
DOI: 10.7554/eLife.68380