scholarly journals A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity

2018 ◽  
Author(s):  
Jeff Granhøj ◽  
Henrik Dimke ◽  
Per Svenningsen
Keyword(s):  
Fluorescent Protein ◽  
Peptide Sequence ◽  
Display System ◽  
Bacterial Display ◽  
Peptide Specificity ◽  
Biotin Ligase ◽  
Novel Variants ◽  
Effective Selection ◽  
Selection Step ◽  
Site Selective

AbstractBiotinylation creates a sensitive and specific tag for purification and detection of target proteins. TheE. coliprotein-biotin ligase BirA biotinylates a lysine within a synthetic biotin acceptor peptide (AP) and allow for specific tagging of proteins fused to the AP. The approach is not applicable to unmodified proteins, and we sought to develop an effective selection system that could form the basis for directed evolution of novel BirA variants with specificity towards unmodified proteins. The system was based on bacterial display of a target peptide sequence, which could be biotinylated by cytosolic BirA variants before being displayed on the surface. In a model selection, the bacterial display system accomplished >1.000.000 enrichment in a single selection step. A randomly mutated BirA library was used to identify novel variants. Bacteria displaying peptide sequences from 13 out of 14 tested proteins were strongly enriched after 3–5 selection rounds. Moreover, a clone selected for biotinylation of a C-terminal peptide from red-fluorescent protein TagRFP showed site-specific biotinylation. Thus, active BirA variants with novel specificity are effectively isolated with our bacterial display system and provides a basis for the development of BirA variants for site-selective biotinylation.

scholarly journals Genetic Transformation of the Obligate Parasite Plasmodiophora brassicae

2013 ◽  
Vol 103 (10) ◽  
pp. 1052-1057 ◽  
Author(s):  
J. Feng ◽  
Sheau-Fang Hwang ◽  
S. E. Strelkov
Keyword(s):  
Genetic Transformation ◽  
Fluorescent Protein ◽  
Plasmodiophora Brassicae ◽  
Expression Vectors ◽  
Obligate Parasite ◽  
Putative Transformants ◽  
Resting Spores ◽  
Selection Step ◽  
Protoplast Preparation ◽  
Green Fluorescent

A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem.

scholarly journals Gene manipulation of fluorescent protein through site-selective hydrolysis by Ce(IV)/EDTA

2005 ◽  
Vol 49 (1) ◽  
pp. 281-282
Author(s):  
Yoshihito Kitamura ◽  
Satoshi Mori ◽  
Makoto Komiyama
Keyword(s):  
Fluorescent Protein ◽  
Selective Hydrolysis ◽  
Gene Manipulation ◽  
Site Selective

scholarly journals Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

2010 ◽  
Vol 76 (8) ◽  
pp. 2410-2418 ◽  
Author(s):  
Shumin Hu ◽  
Jian Kong ◽  
Wentao Kong ◽  
Tingting Guo ◽  
Mingjie Ji
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Surface Display ◽  
Lactobacillus Fermentum ◽  
Display System ◽  
C Terminus ◽  
Bacteriophage Endolysin

ABSTRACT The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.

scholarly journals Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

2012 ◽  
Vol 78 (9) ◽  
pp. 3051-3058 ◽  
Author(s):  
Hyeok-Jin Ko ◽  
Eunhye Park ◽  
Joseph Song ◽  
Taek Ho Yang ◽  
Hee Jong Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Cell Surface Display ◽  
Heterologous Proteins ◽  
Display System ◽  
Reporter Protein ◽  
Content Type ◽  
Ligation Independent Cloning

ABSTRACTAutotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) ofEscherichia colifor the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes fromSaccharophagus degradans2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 104molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growingE. coli.

Site-selective immobilization of streptavidin on enzymatically biotinylated bacterial magnetic particles

2008 ◽  
Vol 1094 ◽  
Author(s):  
Yoshiaki Maeda ◽  
Tomoko Yoshino ◽  
Haruko Takeyama ◽  
Masaaki Takahashi ◽  
Harumi Ginya ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Functional Materials ◽  
Fluorescence Analysis ◽  
Bacterial Cells ◽  
Conjugation System ◽  
Bacterial Magnetic Particles ◽  
Site Selective

AbstractBiotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) on the surfaces of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. Both BAP and green fluorescent protein (GFP) were fused to Mms13 that was isolated from BacMP membranes. The localization of the fusion protein, BAP-Mms13-GFP, was confirmed by fluorescence analysis. BacMPs that expressed BAP-Mms13-GFP (BAP/GFP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. The in vitro biotinylation of BAP/GFP-BacMPs was confirmed using alkaline phosphatase (ALP)-streptavidin detection. The conjugation system developed in this study provides a method for producing biotin- or streptavidin-labeled magnetic nanoparticles without the use of a crosslinker reagent. Various functional materials can be immobilized site-selectively onto these uniquely designed BacMPs. By combining this site-selective biotinylation technology and protein display methodology, increasingly innovative and attractive magnetic nano-materials can be constructed.

scholarly journals Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869

2006 ◽  
Vol 72 (11) ◽  
pp. 7183-7192 ◽  
Author(s):  
Yoshimi Kikuchi ◽  
Masayo Date ◽  
Hiroshi Itaya ◽  
Kazuhiko Matsui ◽  
Long-Fei Wu
Keyword(s):  
Corynebacterium Glutamicum ◽  
Signal Peptide ◽  
Fluorescent Protein ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Gram Negative ◽  
Tat Pathway ◽  
Tat System ◽  
Folded Proteins ◽  
Green Fluorescent

ABSTRACT Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.

scholarly journals Computational Elucidation of Recombinant Fusion Protein Effect on Peptide-Directed Nanoparticles

2021 ◽  
Author(s):  
Adithya Polasa ◽  
Imann Mosleh ◽  
James Losey ◽  
Alireza Abbaspourrad ◽  
Robert Beitle ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Peptide Sequence ◽  
Average Particle ◽  
Binding Affinities ◽  
Free Energies ◽  
Free Peptide ◽  
In The Wild ◽  
Energy Perturbation ◽  
Dynamics Simulations ◽  
Green Fluorescent

AbstractNanoparticles synthesized using various peptides have optimized properties and functional abilities which can be achieved via peptide flexibility and site specificity. Using peptide Pd4 and other alanine substitution combinations of Pd4 attached to a green fluorescent protein (GFPuv), nanoparticles with well-defined sizes that are soluble in aqueous solutions can be produced. In this study, extensive molecular dynamics simulations explored the structural and functional differences between the free peptides and the peptides bound to the GFPuv used in nanoparticle production. Binding affinities of histidines of Pd4 peptide and its two mutants A6 and A11 to a palladium atom were calculated using the free energy perturbation method. Interestingly, the average particle sizes obtained from transmission electron microscopy (TEM) images correlated with our calculated free energies of different peptide sequences. Remarkably, when the peptide was bound to GFPuv, the free energies of histidine were very similar in the wild-type and other mutated peptides. However, this trend is not observed with free peptide simulations, where binding affinities differ by mutation of histidine residues. This study describes, at a molecular level, the role of amino acid sequence on binding affinity of the peptide to the surface of the palladium particles, and the functional ability of the GFPuv protein controlling these free energies irrespective of peptide sequence. Our study will provide a framework for designing free and protein attached peptides that facilitate peptide-mediated nanoparticle formation with well-regulated properties.

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