Expressed protein ligation. Method and applications.
The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.
David
Ralf
R
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
Richter
Michael P O
MP
Beck-Sickinger
Annette G
AG
D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D019154:Protein Splicing; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004952:Esters; D010446:Peptide Fragments; D011506:Proteins; D013438:Sulfhydryl Compounds; D010450:Endopeptidases; D003545:Cysteine