title: Bacterial expression and/or solid phase peptide synthesis of 20-40 amino acid long polypeptides and miniproteins, the case study of Class B GPCR ligands.
abstract: By using two different synthetic techniques several polypeptides interacting with Class B type G-protein coupled receptors were prepared. These polypeptides of different lengths (20 ≤ amino acids ≤ 40), structural and aggregation properties, were prepared both by solid phase peptide synthesis (SPPS) and E.coli bacterial expression. Their purity, synthetic yields, by-products and (15)N/(13)Clabelling characteristics were compared as function of i) the applied method, ii) amino acid length and iii) folding propensities. Their tentative yields, costs and "environmental footprints" were analyzed and found as follows. For unlabelled and short polypeptides (n= 20 aa.) the method of choice is the less environmentally friendly however, quick and effective SPPS. If the polypeptide is (un)folded and/or has no aggregation propensity, then SPPS gives relatively good yield (e.g. 14 ± 4%) and a pure product (>97%). For aggregating polypeptides production yields drop for both methods 4 ± 2% (SPPS) and 2 ± 1% (E. coli), respectively. For longer (n≥ 30 aa.) macromolecules (e.g. miniproteins) bacterial expression efficacy gets higher. Moreover biotechnology is "greener", the resulting in raw material is purer (2.8 ± 1.5 mg). All these advantages for at a lower cost: ~4 €/aa. If isotopic labelling is needed for heteronuclear NMR measurements, bacterial expression is the sole option, due to the high cost of (15)N/(13)C labelled Fmoc(Boc)-L-aa-OH starting materials needed for SPPS. In E.coli uniformly double-labelled, pure polypeptides can be obtained for less than 5-700 €/mg, regardless of the length of the polypeptide chain. Thus, chemists are encouraged to use E.coli expression systems when adequate to make not only proteins but polypeptides and miniproteins as well.
authors.forename: Pál
authors.initials: P
authors.lastname: Stráner
authors.forename: Nóra
authors.initials: N
authors.lastname: Taricska
authors.forename: Mária
authors.initials: M
authors.lastname: Szabó
authors.forename: Gábor K
authors.initials: GK
authors.lastname: Tóth
authors.affiliation: Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány P. sétány 1/A, Budapest, H-1117 Hungary. perczel@chem.elte.hu.
authors.forename: András
authors.initials: A
authors.lastname: Perczel
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D060327:Solid-Phase Synthesis Techniques / Q000379:methods*
chemical_list: D000596:Amino Acids; D008024:Ligands; D010455:Peptides; D043562:Receptors, G-Protein-Coupled