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SUMMARY:Contr. talk - Small-scale expression and purification of the cytok
 ine receptor\, MPL\, using the ALiCE® cell-free system
DTSTART;VALUE=DATE-TIME:20210526T093000Z
DTEND;VALUE=DATE-TIME:20210526T095000Z
DTSTAMP;VALUE=DATE-TIME:20260721T193236Z
UID:indico-contribution-186-1141@lindico453.srv.lu.se
DESCRIPTION:Speakers: Julie Tucker (University of York)\nMPL (also called 
 TpoR) is the receptor for the haematopoietic cytokine\, thrombopoietin (TP
 O). Together\, MPL and TPO control the production of platelets and the mai
 ntenance of haematopoietic stem cells. Gain of function mutations in MPL c
 onstitute ˜5-10 % of driver mutations in essential\nthrombocythemia and p
 rimary myelofibrosis\, whilst loss of function mutations give rise to thro
 mbocytopenias. MPL comprises an ˜500 aa extracellular domain\, a single t
 ransmembrane helix\, and an ˜120 aa intracellular domain. The extracellul
 ar domain contains the thrombopoietin binding site\, whilst the intracellu
 lar domain provides the binding site for Janus kinase 2\, as well as other
  downstream signalling molecules\, including the Signal Transducers and Ac
 tivators of Transcription\n(STAT) proteins. Structural information on MPL 
 is lacking\, in part due to the challenges of producing recombinant protei
 n. Endogenous expression levels of MPL are very low (< 5000\nreceptors/cel
 l) and heterologous expression of full-length MPL has not been reported. H
 ere we report small-scale expression and purification of both full-length 
 and ectodomain of MPL using the ALiCE® cell-free expression system (Lenio
 Bio GmbH). This plant cell-based system allows for the expression of membr
 ane and secreted proteins within the microsomal compartment\, ensuring the
 ir correct folding\, membrane insertion and glycosylation. Optimisation of
  expression time\, plasmid\nconcentration and downstream processing allowe
 d for production and purification of glycosylated MPL\, the functional cha
 racterisation of which is ongoing.\n\nhttps://lindico453.srv.lu.se/event/2
 19/contributions/1141/
LOCATION:
URL:https://lindico453.srv.lu.se/event/219/contributions/1141/
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SUMMARY:Contr. talk - From expression to purified protein: a P4-ATPase for
  CryoEM studies
DTSTART;VALUE=DATE-TIME:20210526T091000Z
DTEND;VALUE=DATE-TIME:20210526T093000Z
DTSTAMP;VALUE=DATE-TIME:20260721T193236Z
UID:indico-contribution-186-1140@lindico453.srv.lu.se
DESCRIPTION:Speakers: LineMarie Christiansen (Aarhus University)\nThe Sacc
 haromyces cerevisiae P4-ATPase Neo1p is believed to function as a lipid fl
 ippase\, translocating lipids towards the cytosolic leaflet of both Golgi 
 and endosome membranes. In\norder to screen around both functional and str
 uctural studies to ascertain the exact role and function of Neo1p an effic
 ient high-yield expression and purification protocol is vital. By\noverexp
 ression in S. cerevisiae and purification based on a high-specificity tag 
 we are left with a pure protein sample ideal for both structural and funct
 ional studies. For CryoEM\ndetergent-exchange into LMNG can be easily perf
 ormed\, with some attention to detergent concentration\n\nhttps://lindico4
 53.srv.lu.se/event/219/contributions/1140/
LOCATION:
URL:https://lindico453.srv.lu.se/event/219/contributions/1140/
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SUMMARY:Keynote talk - Whipping GPCRs into shape for structure determinati
 on
DTSTART;VALUE=DATE-TIME:20210526T083000Z
DTEND;VALUE=DATE-TIME:20210526T091000Z
DTSTAMP;VALUE=DATE-TIME:20260721T193236Z
UID:indico-contribution-186-1139@lindico453.srv.lu.se
DESCRIPTION:Speakers: Christopher G. Tate (MRC Laboratory of Molecular Bio
 logy\, UK)\nOver the past 15 years we have determined multiple structures 
 of GPCRs by both X-ray crystallography and cryo-EM. For each GPCR we had t
 o overcome the difficulties of overexpression\, solubilisation\, stabilisa
 tion and structure determination\, as you would for any membrane protein. 
 For X-ray crystallography\, extensive protein engineering was required to 
 form well-diffracting crystals\, in parallel with judicious choices of det
 ergent\, ligand and crystallisation format (vapour diffusion or lipidic cu
 bic phase). Different strategies also had to be devised depending on which
  conformational state was required for structure determination. The advent
  of high-resolution structure determination by single particle cryo-EM has
  presented new opportunities for GPCR structural biology\, and structures 
 are now being determined that would have been onerous\, if not impossible\
 , to determine by X-ray crystallography. I will present the key factors fo
 r expression\, purification and structure\ndetermination that have made GP
 CR structures possible.\n\nhttps://lindico453.srv.lu.se/event/219/contribu
 tions/1139/
LOCATION:
URL:https://lindico453.srv.lu.se/event/219/contributions/1139/
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