The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins.
Mini-inteins derived from Synechocystis sp. (Ssp DnaB intein) and Mycobacterium xenopi (Mxe GyrA intein) that have been modified to cleave peptide bonds at their C and N termini, respectively, were cloned in-frame to the N and C termini of a target protein. Peptide bond cleavage of the modified inteins generated an N-terminal cysteine and a C-terminal thioester on the same protein. These complementary reactive groups underwent intra- or intermolecular condensation to generate circular or polymeric protein species with a new peptide bond at the site of ligation. Three cyclic peptides, BBP, an organ specific localization peptide; RGD, an inhibitor of platelet aggregation; and CDR-H3/C2, which inhibits HIV-1 replication, were isolated using the two-intein system. BBP, RGD, and CDR-H3/C2 had masses of 977.1, 1119.9, and 2098.6 g/mol, respectively, as determined by matrix-assisted laser desorption-time of flight mass spectrometry, which agreed well with the values of 977.2, 1120.3, and 2098.3 g/mol, respectively, predicted for the cyclic species. This system was used to cyclize proteins as large as 395 amino acids. Furthermore, multimers of thioredoxin were formed upon concentration of the reactive species, indicating the potential to form novel biomaterials based on fibrous proteins.
Evans
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TC
New England Biolabs, Inc., Beverly, Massachusetts 01915-5599, USA.
Benner
J
J
Xu
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MQ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D000458:Cyanobacteria; D003545:Cysteine / Q000378:metabolism; D027081:DNA Gyrase; D004265:DNA Helicases / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D053488:DnaB Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D019912:Mycobacterium xenopi; D010456:Peptides, Cyclic / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism*; D019154:Protein Splicing
D001426:Bacterial Proteins; D010456:Peptides, Cyclic; D011108:Polymers; D004265:DNA Helicases; D053488:DnaB Helicases; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D003545:Cysteine