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Protein engineering by chemical methods

Ball, Haydn Lewis; (1993) Protein engineering by chemical methods. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Procedures and methodologies for the routine chemical synthesis and purification of proteins is reported. Optimised automated protocols of classical stepwise Merrifield solid phase peptide synthesis (SPPS) have been used to synthesise 99 residue M. tuberculosis and 97 residue E. coli Heat Shock 10kD proteins. Since peptide bond formation in stepwise SPPS is not unequivocable and leads to the generation of a family of chromatographically similar deletion and truncated peptides, a purification system that is independent of sequence length and amino acid composition has been developed. The proposed system is based on the combination of (i) an effective capping protocol after each coupling step and (ii) the addition of a removable protecting group to the N-terminus of the last amino acid, bearing either lipophilic, acidic or basic functions. Purification of the crude cleavage product has been performed on reversed-phase or ion- exchange media, depending on the probe used. After purification the probe molecule is quantitatively removed from the peptide chain through a base catalysed β-elimination reaction to yield the pure homogeneous product. The potential of these chromatographic probes is demonstrated by application to model peptides (linear and cyclic) from 17 to 104 residues in length. An alternative to the chemical synthesis of proteins by stepwise SPPS, is the fragment condensation of fully protected peptide segments. The synthesis of a model peptide, GRF(1-44)+Gly, using solid phase fragment condensation is described. Attention is focused on the use of relatively large protected peptides (15-20 residues) with reference to (i) the most effective linker and therefore synthetic chemistry (Boc vs. Fmoc) for their production, (ii) their purification on a large scale and (iii) their coupling to a resin-bound peptide fragment. The most effective protocol is to synthesise the 15 and 17 residue fragments on Sasrin resin (Fmoc chemistry) and to purify them in large quantities using perfusion chromatography. Solid phase fragment condensation is performed in DMSO/NMP, using DCC/HOBt activation, with overall yield of 50%.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Protein engineering by chemical methods
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10106967
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