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

Stráner, Pál and Taricska, Nóra and Szabó, Mária and Tóth, Gábor and Perczel, András (2016) 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. Current Protein and Peptide Science, 17. pp. 147-155. ISSN 1389-2037

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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/13C-labelling 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 rela- tively 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) bacte- rial 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/13C 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.

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