A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes

Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6′-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636–655 nm. Among them, the combination of PxRE-R215K mutant with 6′-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.

Download Full-text

Covariance of Complementary rRNA Loop Nucleotides Does Not Necessarily Represent Functional Pseudoknot Formation In Vivo

Journal of Bacteriology ◽

10.1128/jb.182.20.5671-5675.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5671-5675 ◽

Cited By ~ 2

Author(s):

Natalya S. Chernyaeva ◽

Emanuel J. Murgola

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Cell Growth ◽

Site Directed Mutagenesis ◽

23S Rrna ◽

Directed Mutagenesis ◽

Bacterial Cells ◽

Domain I

ABSTRACT We examined mutationally a two-hairpin structure (nucleotides 57 to 70 and 76 to 110) in a region of domain I ofEscherichia coli 23S rRNA that has been implicated in specific functions in protein synthesis by other studies. On the basis of the observed covariance of several nucleotides in each loop inBacteria, Archaea, and chloroplasts, the two hairpins have been proposed to form a pseudoknot. Here, appropriate loop changes were introduced in vitro by site-directed mutagenesis to eliminate any possibility of base pairing between the loops. The bacterial cells containing each cloned mutant rRNA operon were then examined for cell growth, termination codon readthrough, and assembly of the mutant rRNAs into functional ribosomes. The results show that, under the conditions examined, the two hairpins do not form a pseudoknot structure that is required for the functioning of the ribosome in vivo and therefore that sequence covariance does not necessarily indicate the formation of a functional pseudoknot.

Download Full-text

In vivo elimination of parental clones in general and site-directed mutagenesis

Journal of Immunological Methods ◽

10.1016/j.jim.2014.12.008 ◽

2015 ◽

Vol 417 ◽

pp. 67-75 ◽

Cited By ~ 7

Author(s):

Erika G. Holland ◽

Felicity E. Acca ◽

Kristina M. Belanger ◽

Mary E. Bylo ◽

Brian K. Kay ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis

Download Full-text

The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

Biological Chemistry ◽

10.1515/hsz-2017-0178 ◽

2017 ◽

Vol 399 (1) ◽

pp. 73-77 ◽

Cited By ~ 7

Author(s):

Monika B. Dolinska ◽

Yuri V. Sergeev

Keyword(s):

Oculocutaneous Albinism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Multiple Site ◽

Insect Larvae ◽

Melanin Biosynthesis ◽

Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

Download Full-text

Mending mutations by in vivo site-directed mutagenesis

Molecular Medicine Today ◽

10.1016/s1357-4310(98)01253-2 ◽

1998 ◽

Vol 4 (5) ◽

pp. 188-189

Author(s):

Natasha J Caplen

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis

Download Full-text

Mutant Firefly Luciferase Enzymes Resistant to the Inhibition by Sodium Chloride

10.21203/rs.3.rs-243105/v1 ◽

2021 ◽

Author(s):

Satoshi Yawata ◽

Kenichi Noda ◽

Ai Shimomura ◽

Akio Kuroda

Keyword(s):

Sodium Chloride ◽

Double Mutant ◽

Luciferase Activity ◽

Firefly Luciferase ◽

Activity Level ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Original Activity ◽

Double Mutation

Abstract ObjectivesFirefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of firefly luciferase in the presence of sodium chloride.ResultsWe first obtained two mutant luciferase enzymes whose inhibition were alleviated and identified these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

Download Full-text

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.195-196.407 ◽

2012 ◽

Vol 195-196 ◽

pp. 407-411

Author(s):

Mu Qing Qiu

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Recombinant Plasmid ◽

Genomic Dna ◽

Gene Replacement ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

Download Full-text