An Antibiotic Selection System For Protein Overproducing Bacteria

Introduction: Protein overproduction is a major bottleneck for analyses of membrane proteins and for the construction of cell factories. Screening for optimized protein production can be very time consuming. In this study we show that the coupling of antibiotic resistance to poorly produced membrane proteins of Escherichia coli can be used as a fast and simple selection system for protein overproduction.Methods: We designed an expression plasmid encoding the gene of interest and an additional, inducible antibiotic resistance marker. Both genes were linked by a hairpin structure that translationally couples the genes. Consequently, high expressing gene variants also allow for higher production of the coupled antibiotic resistance marker. Therefore, high expressing gene variants in a library can be determined either by plating the expression library on selection plates or by growing the library in a liquid culture, while both contain high concentrations of the inducible antibiotic.Results: We designed libraries for membrane proteins of E. coli, based on a recently published technique that promises enhanced protein production by optimizing the nucleotides between the Shine Dalgarno sequence and the start codon, an integral part of the translation initiation region. We successfully tested the expression of these libraries with the antibiotic selection system on plates and in liquid cultures.Conclusions: We successfully implemented the antibiotic selection system and confirmed enhanced protein production when applying the above-mentioned optimization technique.