BCH3052 Lecture Notes - Lecture 16: Cyclophilin, Cyclic Peptide, X-Ray Crystallography
25 May 2018
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Lecture 16 – Protein Production for Biophysical Characterisation
Case Study: Cyclosporin from Fungus
• Discovered in 1971
• 1976: immunosuppressive activity was discovered, which changed the face of
organ transplantation
• A peptide like molecular
o Peptide vs. protein:
▪ Peptide: protein chains that are small enough to synthesis (up to
50 aa)
• Cyclosporin A is 11 residue non-ribosomal cyclic peptide
• Cyclic structure makes it less susceptible to digestive enzymes – administered
orally
• Non DNA encoded peptide
• Cyclosporin A acts (CsA) forms a complex with cyclophilin - inhibits
calcineurin → preventing activation of NF-AT (nuclear activator of activated
T cells)
• Study the peptide through NMR and X-ray crystallography
o Structure of CyA bound to cyclophilin was completely different – want
to stabilise or mimic
Production of the Protein – Recombinant DNA Technology
• DNA encoding the desired protein is produced by
restriction site digest or PCR
• Inserted into a plasmid to produce a recombinant
plasmid
• If protein isn’t naturally abundant or not easily
obtained from a biological tissue – produced by
recombinant protein expression
• Recombinant protein expression
o Plasmid is incorporated into a host cell to
make transformed cell → transformed cells selected via
antibiotic resistance conferred by the plasmid
o Cells that grow have taken up plasmid
o Transformed cells can be grown up in culture and then
induced to overexpress the foreign protein – isotopic
labels
• Some proteins don’t get made properly in E. coli
Prokaryotic vs. Eukaryotic Systems
• E. coli:
o Very easy to handle and have well characterised
genetics
o Grow rapidly to high density
o Utilise inexpensive substrates
o Many cloning vectors available BUT
• Sometimes misfold proteins, produce insoluble proteins or proteins are
degraded
• Do not perform posttranslational modifications such as phosphorylation and
glycosylation
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Lecture 16 protein production for biophysical characterisation. Case study: cyclosporin from fungus: discovered in 1971, 1976: immunosuppressive activity was discovered, which changed the face of organ transplantation, a peptide like molecular, peptide vs. protein, peptide: protein chains that are small enough to synthesis (up to. T cells: study the peptide through nmr and x-ray crystallography, structure of cya bound to cyclophilin was completely different want to stabilise or mimic. Production of the protein recombinant dna technology: dna encoding the desired protein is produced by restriction site digest or pcr. Inserted into a plasmid to produce a recombinant plasmid. Eukaryotic expression systems: permit folding, post-translational modification and oligomerization of proteins in manners that are often identical to those that occur in mammalian cells. Protein detection: detecting polypeptides eluting from reverse phase columns absorption of. Affinity chromatography: recombinant protein can be produced as a fusion protein (e. g. with his-tag,
