The multifaceted benefits of protein co-expression in Escherichia coli.
We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3'-5' exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-β-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.
Department of Pharmacy and Biotechnology, University of Bologna; CSGI, Department of Chemistry, University of Firenze.
Alessandra
A
Stefan
Department of Pharmacy and Biotechnology, University of Bologna.
Alessandro
A
Ceccarelli
Department of Pharmacy and Biotechnology, University of Bologna.
Emanuele
E
Conte
Department of Pharmacy and Biotechnology, University of Bologna.
Alejandro
A
Montón Silva
Department of Pharmacy and Biotechnology, University of Bologna; CSGI, Department of Chemistry, University of Firenze; a.hochkoeppler@unibo.it.
Alejandro
A
Hochkoeppler
D004258:DNA Polymerase III / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D004258:DNA Polymerase III