title: Physiological adaptation of the bacterium Lactococcus lactis in response to the production of human CFTR.
abstract: Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that different strategies are needed to overcome low expression yields and toxicity.
authors: Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
authors: Anton
authors: A
authors: Steen
authors: Elena
authors: E
authors: Wiederhold
authors: Tejas
authors: T
authors: Gandhi
authors: Rainer
authors: R
authors: Breitling
authors: Dirk Jan
authors: DJ
authors: Slotboom
mesh_terms: D000222:Adaptation, Physiological; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; C505032:CFTR protein, human; D008565:Membrane Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator