Assembly of protein building blocks using a short synthetic peptide.
Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces.
MRC Laboratory of Molecular Biology, Hills Road, CB2 0QH Cambridge, United Kingdom.
Enrico
E
Ferrari
Mikhail
M
Soloviev
Dhevahi
D
Niranjan
Jason
J
Arsenault
Chunjing
C
Gu
Yvonne
Y
Vallis
John
J
O'Brien
Bazbek
B
Davletov
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013696:Temperature
D010455:Peptides; D011506:Proteins