title: Expression of compstatin in Escherichia coli: incorporation of unnatural amino acids enhances its activity.
abstract: Compstatin, a 13-residue cyclic peptide, is a complement inhibitor that shows therapeutic potential. Several previous approaches have improved the activity of this peptide several-fold. In the present study, we have expressed and purified compstatin from Escherichia coli in an effort to increase its potency and to generate it in high yield in a more economical fashion. An intein-based expression system was used to express compstatin in fusion with chitin-binding domain and Ssp DnaB intein, which were later cleaved from the expressed molecule at room temperature and pH 7.0 to yield pure compstatin in one step. The expressed compstatin showed activity similar to the synthetic compstatin in an ELISA-based assay. The same expression system and purification strategy were used to incorporate three tryptophan analogs, 6-fluoro-tryptophan, 5-hydroxy-tryptophan, and 7-aza-tryptophan, into compstatin. Interestingly, incorporation of 6-fluoro-tryptophan increased the activity three-fold relative to wild-type compstatin; in contrast, incorporation of 5-hydroxy- or 7-aza-tryptophan rendered compstatin less active than the wild-type form.
authors.affiliation: Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Madan
authors.initials: M
authors.lastname: Katragadda
authors.forename: John D
authors.initials: JD
authors.lastname: Lambris
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000235:genetics; D015854:Up-Regulation / Q000502:physiology
chemical_list: D010456:Peptides, Cyclic; C111828:compstatin; D014364:Tryptophan