Pichia pastoris is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon Pyrococcus abyssi.
We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence alpha-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3'-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.
Labarre
Cécile
C
Institut de Génétique et Microbiologie (UMR 8621 CNRS Université Paris XI), Université Paris-Sud, Bât 360, Orsay, 91405, France. cecile.labarre@igmors.u-psud.fr
van Tilbeurgh
Herman
H
Blondeau
Karine
K
D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D043903:Pyrococcus abyssi / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D019843:Archaeal Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins