Synthelis - Biotech blog

Proteoliposomes – ideal model systems to study the structure and function of membrane proteins in semi-native environment by cryogenic electron microscopy

What is the N-Protein ?

The nucleocapsid protein (N protein) of SARS-CoV-2 is a structural protein synthesized by the virus, which plays a key role in the RNA packaging into the nucleocapsid. Its function is to mediate viral assembly through interaction with the viral genome and M protein, helping to increase viral RNA transcription and replication. Characterization studies have shown that N protein is very immunogenic and the antibodies against N protein present higher sensitivity and longer persistence than those against other structural proteins of SARS-CoV. These findings allow to consider N protein as a potential target for vaccine development against SARS-CoV-2.

With the COVID-19 pandemic still spreading across the globe, the fight is on to rapidly develop efficient solutions for diagnosis and treatment. In the past, cell-free protein expression has been used to produce antibodies against other strains of coronavirus. As such, could cell-free systems pose a viable option towards finding a solution against SARS-CoV-2 ?

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…

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.

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.

The open nature of cell-free systems allows the production of protein complexes much more easily than with in vivo systems.

The wide availability and convenience of cell-free kits has led to their increased use over the last few years.