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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Miniprotein Engineering of the Knottin-like Scaffold Min-23-Solid-phase Synthesis and Oxidative Folding StrategiesFrederic Zoller 1 , Christain Hauer 2 , Annette Markert 1,2 ,Uwe Haberkorn 1,2 , and Walter Mier 21 Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, 69120,Heidelberg, Germany; 2Clinic for Nuclear Medicine, University Hospital Heidelberg,D-69120, Heidelberg, GermanyIntroductionMiniprotein scaffolds are currently emerging as novel recognition molecules and for thegeneration of epitope binding motifs for medical applications [1]. Owing to theirextraordinary stable architecture that tolerates multiple amino acid substitutions orinsertions, disufide-rich miniproteins are ideal scaffolds for combinatorial engineering [2].The knottin-like peptide scaffold Min-23 is rationally designed from the plant-occurringcyclotide ecballium elaterium trypsin inhibitor II (EETI-II) (Figure 1). This 23-mer peptideincorporating a cystine-stabilised beta-sheet (CSB) motif frames an autonomous foldingunit. The plasticity of the CSB motif allows the integration of a randomized affinityfunction into the stably folded framework [3]. These characteristics classify Min-23 as apeptide scaffold for lead generation using in vitro screening techniques, such as phage orribosome display [4]. However, engineering of cystine-knotted miniproteins is achallenging process, which requires an efficient solid-phase synthesis and oxidative foldingstrategies.EETI-II H 2 N-GCPRILMRCKQDSDCLAGCVCGPNGFCG-CONH 2cysteine protecting group (Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH)MIN-23 H 2 N-LXRCKQDSDCLAGSVCGPNGFCG-CONH 2norleucinesubstitutionpseudo-prolinedipeptidevariableloopFig. 1. Primary structure of EETI-II and Min-23. Key role amino acids for an optimizedFmoc/tBu-solid phase synthesis are highlighted.OtBuOCO NHHOOCOOCHNOHSOFig. 2. Structure of Fmoc-Asp(OtBu)-Cys(ψ H,DMP pro)-OH. This pseudoproline buildingblock avoids secondary structure formation. Moreover, it was shown to preventaspartimide formation and it acts as orthogonal protecting group for cysteine.Results and DiscussionThe initial solid-phase peptide synthesis of Min-23 revealed a high aggregation during thechain elongation within the helical section. This drawback was overcome usingcommercially available Asp-Ser-pseudoproline. In addition, the tertiary amide backbone ofthe Asp-Cys-pseudoproline building block was shown to prevent aspartimide formation(Figure 2) [5]. This optimization results in a crude peptide purity of 76% compared to only42% using standard SPPS-building blocks. For the subsequent oxidative peptide folding viadisulfide formation two different strategies were developed. The simultaneous formation oftwo disulfide bridges was achieved by air oxidation in a DMSO-free, buffered solution in534