Investigations of combinations of mutations in the jellyfish green fluorescent protein (GFP) that afford brighter fluorescence, and use of a version (VisGreen) in plant, bacterial, and animal cells

Teerawanichpan, Prapapan; Hoffman, Travis; Ashe, Paula; Datla, Raju; Selvaraj, Gopalan

doi:10.1016/j.bbagen.2007.06.005

Cited by 46 publications

(38 citation statements)

References 39 publications

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“…The plasmid pBM22 contained the parFG-mCherry-parH module and a lacO 120 array (37). A plasmid encoding the ParF-Emerald fusion was constructed in two steps: first, parF was cloned in frame with the gene encoding Emerald in plasmid pPT100 (38). Then the parF-emerald fusion gene was amplified by polymerase chain reaction and cloned into pBAD30 under the control of an arabinose-inducible promoter.…”

Section: Methodsmentioning

confidence: 99%

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

et al. 2016

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Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.

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Section: Methodsmentioning

confidence: 99%

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

et al. 2016

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“…We introduced the mutations of superfolder GFP (S30R, Y39N, F64L, F99S, N105T, Y145F, M153T, V163A, I171V, and A206V) into GFP nt -r5M. It was also reported that N149K [28] and S208L [29] affected the folding efficiency of GFP positively, although their effects were not significant. The two mutations (N149K and S208L) were additionally introduced, and the resulting variant was named GFPhs-r5M.…”

Section: Resultsmentioning

confidence: 99%

“…The higher folding efficiency and folding robustness of GFPhs-r5M than those of GFP nt indicates that the superfolder mutations might presumably provide GFP nt -r5M with more stabilization energy than such compensating energy. On the other hand, we presume that the higher specific fluorescence of GFPhs-r5M than GFP nt might be caused by the mutations such as F64L, F99S and N149K mutations which can change the spectral properties of GFP by enhancing the hydrogen bonding networks around the chromophore [22], [26], [28], [29]. Further mutagenesis and structural studies need to be performed to understand the improved folding and spectral properties of the variants more exactly.…”

Section: Discussionmentioning

confidence: 98%

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

et al. 2012

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N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.

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“…A first variant (GFPhs1) was constructed by introducing the superfolder GFP mutations, [19] cycle three mutations, [20] S208L [21] and N149K [22] into GFPcon. The second variant (GFPhs2) was constructed by further addition of nine mutations of GFP variant L024_3-3 [12] into GFPhs1 (Table S1 in the Supporting Information shows the GFPhs1 and GFPhs2 sequences).…”

mentioning

confidence: 99%

Engineering Protein Sequence Composition for Folding Robustness Renders Efficient Noncanonical Amino acid Incorporations

et al. 2010

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Investigations of combinations of mutations in the jellyfish green fluorescent protein (GFP) that afford brighter fluorescence, and use of a version (VisGreen) in plant, bacterial, and animal cells

Cited by 46 publications

References 39 publications

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

Engineering Protein Sequence Composition for Folding Robustness Renders Efficient Noncanonical Amino acid Incorporations

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