Abstract：
: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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Linares DM,Geertsma ER,Poolman Bdoi
10.1016/j.jmb.2010.06.002subject
Has Abstractpub_date
2010-08-06 00:00:00pages
45-55issue
1eissn
0022-2836issn
1089-8638pii
S0022-2836(10)00602-9journal_volume
401pub_type
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