High-throughput mutagenesis using a two-fragment PCR approach

Heydenreich, Franziska M.; Miljuš, Tamara; Jaussi, Rolf; Benoit, Roger; Milić, Dalibor; Veprintsev, Dmitry B.

doi:10.1038/s41598-017-07010-4

Cited by 44 publications

(36 citation statements)

References 28 publications

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“…All truncated AtETR1 constructs and AtETR1 1–307 mutants were prepared in pTEV-16b vector backbone 43 , a modified version of pET-16b (Novagen, Darmstadt, Germany) containing the N-terminal decahistidine-tag followed by a linker (SSGH) and a tobacco etch virus (TEV) protease cleavage site (ENLYFQG; instead of a Factor Xa cleavage site in pET-16b). The new constructs were made by using a two-fragment PCR approach 44 starting from the expression plasmid pTEV-16b-AtETR1 that contains the full-length Arabidopsis thaliana ethylene receptor 1 (AtETR1) cDNA. In short, the mutagenesis PCR primers were designed in either PCRdesign or AAscan program 45 with a 21-nucleotides overlap for a mutagenesis primer pair.…”

Section: Methodsmentioning

confidence: 99%

“…Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment. A pair of fragments was combined into the target plasmid in Gibson assembly 46 , as described in our earlier report 44 . A detailed overview of the molecular cloning as well as the sequences of primers and gene fragments are given in Supplementary Tables S2 – S4 .…”

Section: Methodsmentioning

confidence: 99%

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Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1

Milić

Dick

Mulnaes

et al. 2018

Sci Rep

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Synthetic peptides derived from ethylene-insensitive protein 2 (EIN2), a central regulator of ethylene signalling, were recently shown to delay fruit ripening by interrupting protein–protein interactions in the ethylene signalling pathway. Here, we show that the inhibitory peptide NOP-1 binds to the GAF domain of ETR1 – the prototype of the plant ethylene receptor family. Site-directed mutagenesis and computational studies reveal the peptide interaction site and a plausible molecular mechanism for the ripening inhibition.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1

Milić

Dick

Mulnaes

et al. 2018

Sci Rep

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“…High-throughput, site-directed scanning mutagenesis techniques are widely used in protein engineering. These enable studies of protein structure and function at single residue resolution (1,2,33,34). A deep mutational scanning-based approach, "mutational antigenic profiling" was recently used to map epitopes of nine HIV-1 bNAbs (35).…”

Section: Discussionmentioning

confidence: 99%

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

Datta

Chowdhury

Manjunath

et al. 2020

Preprint

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13Identification of specific epitopes targeted by neutralizing antibodies is essential to advance 14 epitope-based vaccine design strategies. We report a methodology for rapid epitope mapping 15 of neutralizing antibodies against HIV-1 Env at single residue resolution, using viral 16 neutralization assays and deep sequencing. We extended our methodology to map multiple 17 specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as 18 in HIV-We recently described a methodology to decipher epitopes of monoclonal antibody panels 35 using yeast surface display, Cys labeling and deep sequencing 1, 2 . Here we built on this to map 36 epitopes of neutralizing monoclonal antibodies and polyclonal sera against the HIV-1 virus. 37 The design of an effective vaccine against HIV-1 has met with limited success so far, because 38 of the inability of immunogens to elicit broadly neutralizing antibodies (bNAbs) targeted to the 39 trimeric envelope (Env) glycoprotein, the sole viral antigen on the surface of the virus. Most 40 bNAbs isolated from infected individuals are directed to one of the major sites of vulnerability 41 on Env: V1/V2 loop apex, V3 loop, CD4-binding site, centre of the gp120 silent face, gp120-42 gp41 subunit interface, gp41 fusion peptide and membrane proximal external region (MPER) 43 of gp41. 44 We developed a facile methodology for mapping neutralizing HIV-1 epitopes at single residue 45 resolution. To this end, a library of single-site Cys mutations were engineered at selected 46 solvent-exposed sites in the Env glycoprotein on the surface of the virus. Cys mutants were 47 covalently labeled using a Cys reactive reagent (Maleimide-PEG2-Biotin) that masks epitope 48 residues, followed by infection of the labeled mutant viruses in mammalian cells in the 49 presence of bNAbs. Subsequently, deep sequencing of the env gene from bNAb-resistant 50 viruses was used to delineate epitopes ( Figure 1). We focused our epitope mapping efforts to 51 the Env ectodomain of the clade B, CCR5-tropic primary HIV-1 isolate JRFL, which has been 52 extensively characterized both biochemically and structurally, and is used as a model primary 53 virus. 54A total of 81 solvent-exposed residues (Table S1) were selected from the X-ray crystal structure 55 of the pre-fusion HIV-1 Env ectodomain using a combined criterion of >30% solvent 56 accessibility and sidechain-sidechain centroid distance >8 Å ( Figure S1). This led to the 57 identification of a set of solvent-exposed residues which collectively exhibit uniform surface 58 coverage of the Env trimer ( Figure S2). These Env residues were mutated to Cys in the pLAI-59 JRFL molecular clone, pooled in equimolar quantities to create a plasmid DNA library, and 60 transfected in HEK293T cells to produce the Exposed Cys Library, a library of single-site Cys 61 mutant viruses. Wt Env lacks free Cys residues. In single-cycle and multi-cycle infectivity 62 assays, the Exposed Cys Library retained significant viral infectivity ( Figure S3) and ...

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“…Site-directed mutagenesis of the FaJAZ1 degron. Three FaJAZ1 degron mutants and two JAZ1 chimeras of F. × ananassa and A. thaliana were made from JAZ1 orthologs, specifically in the degron sequence IPMQRK and LPIARR using site-directed mutagenesis by PCR 56 . Firstly, primer sequences with muted nucleotides were designed for the construction of FaJAZ1_AK, FaJAZ1_RA, FaJAZ1_AA mutants, and At/FaJAZ1, Fa/AtJAZ1 chimeras ( Supplementary Table S5).…”

Section: Scientific Reports |mentioning

confidence: 99%

A new functional JAZ degron sequence in strawberry JAZ1 revealed by structural and interaction studies on the COI1–JA-Ile/COR–JAZs complexes

Garrido‐Bigotes

Valenzuela-Riffo

Torrejón

et al. 2020

Sci Rep

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The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes as developmental and defense responses. JA-Ile mediates the interaction between the F-box protein COI1 (part of the SCF COI1 E3 ubiquitin ligase) and a JAZ repressor leading to early jasmonate responses. The Arabidopsis JAZ1 protein contains the canonical LPIARR degron sequence, which is responsible for the stabilization of the AtCOI1-JA-Ile-AtJAZ1 complex. In strawberry (Fragaria × ananassa) JAZ family was described at the transcriptional level during fruit development but the information about the interaction mode of this complex is still scarce at the molecular level. To gain insight into the strawberry JA-Ile receptor complex, we evaluated the interaction at the structural level, and protein models were built and analyzed for FaCOI1 and FaJAZ1, FaJAZ8.1, and FaJAZ10. The interaction between FaCOI1 and FaJAZ1, FaJAZ8.1 and FaJAZ10 were explored using several ligands, through molecular docking and molecular dynamics (MD) simulations, finding the strongest interaction with (+)-7-iso-JA-Ile than other ligands. Additionally, we tested interactions between FaCOI1 and FaJAZs by yeast two-hybrid assays in the presence of coronatine (COR, a JA-Ile mimic). We detected strong COR-dependent interactions between FaCOI1 and FaJAZ1. Interestingly, FaJAZ1 contains a new non-canonical (IPMQRK) functional degron sequence, in which Arg and Lys are the key residues for maintaining the interaction of the FaCOI1-COR-FaJAZ1 complex as we observed in mutated versions of the FaJAZ1 degron. Phylogenetic analysis showed that the IPMQRK degron is only present in orthologs belonging to the Rosoideae but not in other Rosaceae subfamilies. Together, this study uncovers a new degron sequence in plants, which could be required to make an alternative and functional JA-Ile perception complex in strawberry. Jasmonates (JAs) are phytohormones that regulate development and stress processes in land plants 1-3. The (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile) is the bioactive jasmonate in Arabidopsis 4 and accumulates at the early stages of development in grape and strawberry fruits 5,6. The bacterial phytotoxin coronatine (COR) is structurally and functionally analogous to JA-Ile 7 .

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High-throughput mutagenesis using a two-fragment PCR approach

Cited by 44 publications

References 28 publications

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

A new functional JAZ degron sequence in strawberry JAZ1 revealed by structural and interaction studies on the COI1–JA-Ile/COR–JAZs complexes

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