Evolved Lactococcus lactis strains for enhanced expression of recombinant membrane proteins.
The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant membrane proteins. By fusing green fluorescent protein and an erythromycin resistance marker (ErmC) to the C-terminus of a target protein, one simultaneously selects for variants with enhanced expression (increased erythromycin resistance) and correct folding (green fluorescent protein fluorescence). Three evolved hosts, displaying 2- to 8-fold increased expression of a plethora of proteins, were fully sequenced and shown to carry single-site mutations in the nisK gene. NisK is the sensor protein of a two-component regulatory system that directs nisin-A-mediated expression. The levels of recombinant membrane proteins were increased in the evolved strains, and in some cases their folding states were improved. The generality and simplicity of our approach allow rapid improvements of protein production yields by directed evolution in a high-throughput way.
Linares
Daniel M
DM
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Geertsma
Eric R
ER
Poolman
Bert
B
D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics / Q000502:physiology; D017930:Genes, Reporter; D016680:Genome, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D030781:Organisms, Genetically Modified; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D015854:Up-Regulation / Q000235:genetics
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins