scholarly journals Simple Preparation of Green Fluorescent Protein Conjugated with ^|^beta;-Cyclodextrin in a Site Specific Manner

2013 ◽  
Vol 29 (8) ◽  
pp. 811-814 ◽  
Author(s):  
Miho SUZUKI ◽  
Yoshihiro ISHIMARU ◽  
Ayumu SAITO ◽  
Koichi NISHIGAKI
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Site Specific ◽  
Beta Cyclodextrin ◽  
Simple Preparation ◽  
Specific Manner ◽  
Green Fluorescent ◽  
A Site

Optimization of the posttranslational click modification of proteins

2011 ◽  
Vol 76 (9) ◽  
pp. 1089-1101
Author(s):  
Milan Vrabel ◽  
Emine Kaya ◽  
Stefan Prill ◽  
Veronika Ehmke ◽  
Thomas Carell
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Fluorescent Protein ◽  
Click Reaction ◽  
Yellow Fluorescent Protein ◽  
Trna Synthetase ◽  
Site Specific ◽  
Specific Manner ◽  
Modified Proteins ◽  
A Site

In order to develop efficient methods that would enable the synthesis of posttranslationaly modified proteins in a site-specific manner we have adopted the orthogonal pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode various pyrrolysine analogs, which we were able to insert into the yellow fluorescent protein (YFP). These experiments showed that the alkene and alkyne containing amino acids 5 and 6 are superior substrates for the pyrrolysyl-tRNA synthetase and that they can be successfully incorporated into proteins. Using the Cu(I)-catalyzed Huisgen–Meldal–Sharpless click reaction, the alkyne containing YFP was finally glycosylated with various sugars. We confirmed the presence of the modified amino acids as well as the corresponding sugar modifications by HPLC-MS/MS mass spectrometry.

scholarly journals Green fluorescent protein gene for site-specific integration into the locus of the rabbit whey acidic protein gene

2020 ◽  
Author(s):  
V.A. Ezerskii ◽  
◽  
E.M. Koloskova ◽  
T.P. Trubitsina ◽  
◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Whey Acidic Protein ◽  
Acidic Protein ◽  
Green Fluorescent Protein Gene ◽  
Site Specific ◽  
Site Specific Integration ◽  
Green Fluorescent ◽  
Rabbit Milk

The high content of whey acidic protein in rabbit milk makes the gene of this protein a promising candidate for its replacement by the gene of pharmacologically active protein using the CRISPR/Cas9 system. The plasmid that contains 5’ and 3’ arms of homology to the rabbit WAP gene was created. A fragment containing a green fluorescent protein gene under the CMV promoter has been integrated into this site. A strategy of making double-stranded cuts in the gene WAP and receiving four pX330 plasmids encoding the endonuclease Cas9 and guide RNAs was developed. The plasmid containing a fragment cmvEGFP was designed for site-specific integration by homologous recombination into the gene WAP to assess the effectiveness of site-specificity of components of the CRISPR/Саѕ9 in vitro.

215 LIVE PIGLETS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER FOLLOWING TARGETING OF A PORCINE ENHANCED GREEN FLUORESCENT PROTEIN LINE MEDIATED BY ZINC-FINGER NUCLEASES TO ESTABLISH CLONED HYGROMYCIN-RESISTANT PRIMARY CELL LINES SUITABLE FOR Cre-MEDIATED RECOMBINASE-MEDIATED CASSETTE EXCHANGE

2014 ◽  
Vol 26 (1) ◽  
pp. 221 ◽  
Author(s):  
A. Perota ◽  
I. Lagutina ◽  
R. Duchi ◽  
P. Turini ◽  
G. Crotti ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Targeting ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Zinc Finger Nucleases ◽  
Site Specific ◽  
Cassette Exchange ◽  
Hygromycin Selection ◽  
Green Fluorescent ◽  
Targeting Vector

Recently, site-specific nucleases (zinc-finger nucleases, ZFN; TAL effector nucleases; and CRISPR) emerged as powerful tools for gene modification of different cells types and enhanced green fluorescent protein (EGFP)-specific ZFN were successfully used in the rat (Geurtz et al. 2010) and in the pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008 Clon. Stem Cells), we generated an EGFP transgenic porcine line (Verro2GFP) characterised by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission, and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated recombinase-mediated cassette exchange (RMCE), using EGFP-specific ZFN. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment = left-homology-arm = LHA; polyA sequence = right-homology-arm = RHA). Cloning floxed (lox2272/lox5171) hygromycin resistance coding sequence between LHA and RHA sequences, we generated the targeting/RMCE vector (pB5′3′Hygro-PL) and its positive control (C+) for PCR set-up (100–1000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM+M199(1 : 1) + 10% FCS, bFGF in 5% CO2, 5% O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2 μg of each ZFN coding vector (Sigma-CompoZr®) and 2 μg of pB5′3′Hygro-PL/KpnI vector were used to “nucleofect” 1.4 × 106 Verro2GFP fibroblasts in 2 experiments. Transfected cells were plated in 20 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 μg mL–1) for 15 days. After 12 days of drug selection, 82 resistant colonies were picked up and expanded in 24 multiwell plates for SCNT. All colonies were PCR screened and 45 (54.9%) colonies were positive. Four colonies were used in zona-free SCNT experiments with 140 Day 6 compacted morulae/blastocysts transferred into 2 synchronized sows that both became pregnant. One pregnancy went to term and delivered 5 live animals and 5 stillborn with correct hygromycin cassette integration, detected by PCR. The PCR products were sequenced in 7 animals to verify integration of promoterless targeting vector and in all 7 sequenced samples we obtained a correct insertion without any substitution/deletion. Using hygromycin selection in these experiments, we demonstrated that ZFN-mediated gene targeting can be easily done with high efficiency and is compatible with living animals. Moreover, we have validated a feasible SCNT-tested platform for further Cre-mediated site-specific gene modifications. This work is supported by a grant (Superpig) co-financed by Lombardy Region through the Fund for Promoting Institutional Agreements.

The Green Fluorescent Protein (GFP)

2013 ◽  
Vol 05 (01) ◽  
pp. 101-102
Author(s):  
Phunlerd Piyaraj
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent
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