RESUMO
A synthetic strategy that allows for the site-specific attachment of polymers such as poly(ethylene glycol) (PEG) to protein pharmaceuticals is described. PEG was attached to a 67-amino acid fully synthetic CCL-5 (RANTES) analogue at its GAG binding site both to reduce aggregation and to increase the circulating lifetime. Effective protection of an Aoaa chemoselective linker during peptide assembly, total chemical protein synthesis, and protein folding was achieved with an isopropylidene group. Mild deprotection of the resulting folded synthetic protein and subsequent polymer attachment occur without interference with the native folded structure and activity.
Assuntos
Quimiocina CCL5/análogos & derivados , Quimiocinas CC/química , Oximas/química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Quimiocina CCL5/química , Quimiocina CCL5/farmacologia , Quimiocinas CC/farmacologia , Glicina/química , HIV-1/efeitos dos fármacos , Humanos , Modelos Moleculares , Polietilenoglicóis/química , Dobramento de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A 27-kDa TASP protein, T(5)Msc(103-151), that was derived from the cytoplasmic domain (amino acid residues 103-151) of the MscL ion channel of M. tuberculosis was synthesized by ketoxime-forming chemoselective ligation between a template molecule carrying five pyruvic acid groups, and linear channel peptides carrying one aminooxyacetic acid group. Ketoxime-forming ligation provided for highly efficient assembly of this large totally synthetic protein construct with yields >90% with modest excess (1.5x) of the aminooxy peptide. Formation of the desired TASP molecule was confirmed by SDS-PAGE analysis and MALDI mass spectrometry. The effect of template attachment on the structure of the peptides constituting the TASP was assessed by circular dichroism spectroscopy. Attachment of the peptides to the topological template induces predominantly helical secondary structure, whereas an analogous peptide that did not bear an aminooxy group, MscL(103-151), does not exhibit significant secondary structure at pH 7 and is found to be monomeric in concentrations up to 65 microM. This observation can be explained by entropic destabilization of the unfolded state of T(5)Msc(103-151) due to the attachment to the template and the resulting loss of degrees of freedom. Pyruvic acid-based ketoxime-forming chemoselective ligation may thus prove to be a useful tool for the assembly of large, non-native protein constructs and their biophysical study.