Pentelute BL.
*
et al.
Massachusetts Institute of Technology, Cambridge, USA
Synthesis of Proteins by Automated Flow Chemistry.
Science 2020;
368: 980-987
Key words
automated synthesis - protein synthesis - barstar
Significance
Solid-phase peptide synthesis (SPPS) is routinely performed for the preparation of
small peptides. Despite decades of optimization, synthesis of peptides with sequences
longer than 50 amino acids is challenging due to the generation of byproducts stemming
from deletion, epimerization, and truncation. The chemical synthesis of full-length
proteins did not become a practical reality until the introduction of native chemical
ligation (NCL), which often requires conditions specific to the protein. Pentelute
and co-workers report an optimized protocol using their automated fast-flow peptide
synthesis (AFPS) system that is capable of synthesizing full-length proteins of up
to 164 amino acids, without the use of chemical ligation techniques, on time-scales
that rival recombinant expression.
Comment
To synthesize full-length proteins, each step in the peptide coupling reaction had
to be carefully optimized. The critical variables the authors identified included
preactivation temperature and time, reaction temperature and Fmoc-deprotection conditions.
The reaction coupling efficiency could be indirectly evaluated using an in-line UV/Vis
spectrometer. AFPS was capable of synthesizing full-length proteins 10 times faster
than traditional SPPS, with higher overall yield and purity. After purification and
folding, the synthesized proteins possessed the same biological activity and biophysical
characteristics as recombinant proteins. By using this technology, unnatural AAs can
be easily incorporated and site-specific mutations are possible.
