Isotope labelling has been used for over 40 years to facilitate the study of protein structure using nuclear magnetic resonance (NMR) spectroscopy. During that time, traditional expression systems such as in vivo protein synthesis were the preferred options for fabricating the labelled proteins. However, nearly a decade ago, it was discovered that cell-free protein expression offers an abundance of beneficial properties making it more adapted to the addition of isotopes than first thought. Here, we give you our top five reasons to use a cell-free system to label your proteins for your NMR study…
Five reasons to label your proteins for NMR studies using cell-free systems.
Topics: Cell-free technology, Proteins
Which extract is best for cell-free protein expression: wheat germ or E. coli?
Cell-free protein expression offers a rapid, reliable technique for synthesis of proteins. Successfully synthesizing large quantities of high-quality product requires finding the optimal protein expression conditions, such as the type of cellular extract used. Lysate choice for the cell- free reaction can affect expression feasibility, yield and cost. Which extract gives the best results? Here, we compare two of the most common: E. coli and wheat germ.
Topics: Cell-free technology, Biotech
Mythbusting #10 : “Cell-free systems are only relevant for protein synthesis"
Cell-free systems are mainly used for in-vitro protein synthesis and can be considered as a powerful tool in genetic code reprogramming, involving the amber codon. In this use, the non-proteinogenic amino-acids are incorporated into proteins by charging them to suppressor-tRNAs molecules that reprogram the existing codons. However, cell-free systems are also used to engineer genetic circuits with applications for in-vitro biology or metabolic engineering.
Topics: Cell-free technology, Proteins, Biotech
Mythbusting #9 : “Cell-free systems do not produce functional protein complexes"
The open nature of cell-free systems allows the production of protein complexes much more easily than with in vivo systems.
Topics: Cell-free technology
Mythbusting #8 : “Cell-free systems are complex; hence the increased uptake in recent years of cell-free kits.”
The wide availability and convenience of cell-free kits has led to their increased use over the last few years.
Topics: Cell-free technology
Mythbusting #7 : “Cell-free systems only work with plasmids”
Topics: Cell-free technology
Cell-free protein synthesis systems: the best chance for your project to succeed !
Topics: Cell-free technology
Mythbusting #6 : “Cell-free systems are not compatible with Good Manufacturing Practices (GMP grade).”
Topics: Cell-free technology
Mythbusting #5 : “Cell-free systems deliver low expression yields.”
Topics: Cell-free technology
Mythbusting #4 : “Cell-free systems only use bacterial extract.”
Escherichia coli (E. Coli) is the most popular cell extract source for cell-free expression systems. However, extracts from wheat germ, rabbit reticulocytes or insect cells are also commonly used. Other mammalian cell lines are also more recently used for cell-free protein biosynthesis. Since they present different properties and advantages, the source of cell extract will be selected according to the properties of the protein to synthesize, such as the need for post-translational modifications, and other constraints, like yield, costs or convenience of cell extract preparation (Carlson et al., 2012; Casteleijn et al., 2013, Zemella et al., 2015).
Topics: Cell-free technology