A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli.
The increasing use of peptides as pharmaceutical agents, especially in the antiviral and anti-infective therapeutic areas, requires cost-effective production on a large scale. Many peptides need carboxy amidation for full activity or prolonged bioavailability. However, this modification is not possible in prokaryotes and must be done using recombinant enzymes or by expression in transgenic milk. Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is suitable for making complex disulfide-containing peptides required in large quantity. Here we describe a method for making amidated peptides using a modified self-cleaving vacuolar membrane ATPase (VMA) intein expression system. This system is suitable for making amidated peptides at a laboratory scale using readily available constructs and reagents. Further improvements are possible, such as reducing the size of the intein to improve the peptide yields (the VMA intein comprises 454 amino acids) and, if necessary, secreting the fusion protein to ensure correct N-terminal processing to the peptide. With such developments, this method could form the basis of a large-scale cost-effective system for the bulk production of amidated peptides without the use of recombinant enzymes or the need to cleave fusion proteins.
Cottingham
I R
IR
PPL Therapeutics (Scotland) Ltd., Roslin Biocentre, Edinburgh EH25 9PP, Scotland. cottingham@ppl-therapeutics.com
Millar
A
A
Emslie
E
E
Colman
A
A
Schnieke
A E
AE
McKee
C
C
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases
D000577:Amides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases