In the present work a modified version of yellow fluorescent protein containing an unnatural structural homologue of the natural amino acid pyrrolysine with a norbornene moiety was produced by expression in Escherichia coli. The incorporation of the unnatural amino acid was achieved by amber stop codon suppression method. A bio-othogonal click reaction was performed, binding a synthetic fluorescent dye to the modified protein. All steps towards necessary for obtaining the genetically modified organism were performed and documented. The artificial amino acid, as well as the dye used in the click reaction were synthetically prepared. The success of the project was demonstrated by LC/MS studies of the products. Fluorescence spectroscopy of click reaction product and the protein was performed, but no conclusive proof of FRET effects could as yet be made. This point remains of interest for future studies.
Table of contents
1. Introduction: Oligonucleotide Synthesis
2. Objective
3. Procedures and Materials
3.1 Chemistry
3.1.1 Methods and Materials utilized
3.1.2 Syntheses
3.2 Biology
3.2.1 Methods and Materials utilized
3.2.2 Primer Synthesis
3.2.3 Plasmid Mini Prep
3.2.4 Agarose Gel Electrophoresis
3.2.5 Polymerase Chain Reaction
3.2.6 Ligation
3.2.7 Transformation
3.2.8 Colony PCR
3.2.9 Protein Expression
3.2.10 Protein Purification
3.2.11 SDS-PAGE
3.2.12 Protein Digest
3.2.13 Protein Click Reaction
4. Results and Discussion
4.1 Agarose Gel Electrophoresis
4.2 SDS PAGE
4.3 Fluorescence Spectroscopy
4.4 Mass Spectrometry
4.4.1 MALDI-TOF
4.4.2 LC / MS
Objective and Research Themes
The goal of this study is to achieve the bacterial expression of a modified yellow fluorescent protein (YFP) through the incorporation of an unnatural amino acid using amber stop codon suppression, followed by a bio-orthogonal click reaction with a fluorescent dye to investigate potential FRET effects.
- Incorporation of unnatural amino acids via amber stop codon suppression.
- Application of bio-orthogonal click chemistry for protein labeling.
- Bacterial expression and purification of genetically modified YFP.
- Analytical validation using mass spectrometry and fluorescence spectroscopy.
Excerpt from the Book
3.2.13 Protein Click Reaction
The bio-orthogonal click reaction between amber YFP and Tetrazine-5-TAMRA requires the presence of DMSO. In order to find the appropriate concentration several conditions were tested, as given in table 13.
The reaction was carried out by heating the mixture to 37 °C in an incubator for 4 h. The insoluble excess of the fluorophore was separated by centrifugation and an SDS PAGE was performed. Most Product was obtained using 20 % DMSO, thus a large-scale reaction of 25 nmol YFP was carried out at this concentration. This assay was heated to 37 °C overnight and after centrifugation the residual dye was removed by Micro Bio-Spin 30 chromatography. This is based on the principle that the relatively small dye molecules can be retained within the pores of polyacrylamide particles whereas the protein is too large to fit into these. The protein-contain fractions were combined and concentrated.
As a control click reaction with compound 4, a fluorescein derivative possessing similar fluorescence behavior as YFP was also carried out in the same manner. Fluorescence spectra of these compounds in aqueous solution were recorded.
Summary of Chapters
1 Introduction: Oligonucleotide Synthesis: Overview of automated solid-state DNA synthesis and the phosphoramidite method used for primer preparation.
2 Objective: Outlines the goal of producing a norbornene-modified YFP for bio-orthogonal click labeling and fluorescence resonance energy transfer studies.
3 Procedures and Materials: Detailed documentation of chemical synthesis protocols, biological expression methods, and analytical preparation techniques.
4 Results and Discussion: Analysis of the experimental outcomes, including PCR validation, protein expression yields, click reaction spectroscopy, and peptide identification via mass spectrometry.
Keywords
Chemical Biology, Unnatural Amino Acid, Amber Stop Codon Suppression, Click Chemistry, YFP, Protein Expression, Mass Spectrometry, Fluorescence Spectroscopy, FRET, Norbornene, Tetrazine, Oligonucleotide Synthesis, Phosphoramidite Method, Protein Purification, SDS-PAGE
Frequently Asked Questions
What is the primary focus of this work?
The work focuses on the genetic modification of yellow fluorescent protein (YFP) by incorporating an unnatural amino acid and subsequently labeling it using bio-orthogonal click chemistry.
What is the central research objective?
The primary objective is to demonstrate the successful site-specific incorporation of a norbornene-functionalized amino acid into YFP and to examine whether this allows for effective fluorescence resonance energy transfer (FRET) after labeling with a rhodamine dye.
Which methods are utilized for protein production?
The study uses amber stop codon suppression for incorporating the unnatural amino acid during bacterial expression in E. coli, followed by purification via his-tag affinity chromatography.
How is the success of the click reaction evaluated?
The success is evaluated through fluorescence spectroscopy to observe spectral changes and mass spectrometry to confirm the presence of the modified peptide fragments.
What analytical techniques are used in the study?
The key techniques include agarose gel electrophoresis for DNA verification, SDS-PAGE for protein purity analysis, mass spectrometry for peptide sequence confirmation, and fluorescence spectroscopy for monitoring labeling effects.
What are the main thematic pillars of the book?
The main themes are synthetic chemistry (amino acid and dye synthesis), molecular biology (cloning and protein expression), and biophysical characterization (spectroscopy and mass spectrometry).
Why was 20% DMSO chosen for the click reaction?
Based on experimental testing of different concentrations, 20% DMSO was found to provide the highest yield for the click reaction between amber YFP and Tetrazine-5-TAMRA.
Was FRET successfully demonstrated?
No, the researchers could not provide conclusive proof of FRET effects from the current data, though they noted a "shoulder" in the fluorescence spectrum at the expected rhodamine wavelength.
Why was mass spectrometry analysis unsuccessful for the whole protein?
The researchers concluded that the protein and its peptides were not suitable for MALDI ionization, as they could not be detected in the analyzer.
- Citation du texte
- Anonym (Auteur), 2014, Unnatural Amino Acid Incorporation and Click Chemistry, Munich, GRIN Verlag, https://www.grin.com/document/416795
