Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

Abstract

Background: Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored.

Results: We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size - all antibiotic-resistant non-producers - was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented.

Conclusion: The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

Figure 1

Small scale expression tests of individual clones in a lacI^q+ or lacI^q- background. (A) Expression test of 10 clones each of the DARPins E3_5 and G3 in E. coli RV308 (lacI^q-) from expression vector pMPAG77 (lacI^q-). MW, molecular weight marker; U, uninduced, number, clone number induced with IPTG. No protein band of the correct size in 15% SDS-PAGE analysis could be observed. Instead, a prominent band of ~19 kDa of a host protein was observed upon induction with IPTG (black arrow), which was identified as E. coli Dps. (B) Expression of DARPins E3_5 and G3 (two individual clones each) in E. coli DH5α (lacI^q-) can only be observed in 15% SDS-PAGE analysis if LacI is provided by the expression plasmid. U, uninduced; I, induced with IPTG.

Figure 2

Sequence Analysis. Sequence of the promoter/operator region, identical in pMPAG6 (lacI^q+) and pMPAG77 (lacI^q-) (top line), and alignment with the deletions (bottom line) isolated from lacI^q-E. coli strains harboring the respective lacI^q- expression vector: identical sequences were found in at least 6 independent clones of each construct protein. The -35 and -10 regions are indicated by yellow boxes; the two lac O1 operator sites are marked by red letters (length defined as the LacI contact residues seen by NMR [36], with each symmetry center in bold). The ribosomal binding site (RBS) is highlighted in green and the start codon in light blue, respectively.