A self-inducible heterologous protein expression system in Escherichia coli

Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. Escherichia coli is a versatile bacterium that has been recognized by drug regulatory authorities and grows rapidly to a high cell density on inexpensive carbon sources. E. coli is the host of choice for the first attempt at recombinant protein production, regardless of the original source 1-4. One of the most commonly used E. coli expression systems relies on the inducible T7 RNA polymerase because this system obtains high yields of recombinant proteins 5,6. The coding sequence of the T7 RNA polymerase is inserted into the bacterial chromosome under the control of the inducible lac UV5 operon and is transcribed by the endogenous E. coli polymerase. The lac repressor protein (LacI) regulates access to the T7 RNA polymerase coding sequence by binding to the lac UV5 operon. Protein expression induction is triggered by the addition of the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG), which is a structural non-metabolizable analogue of allolactose. The T7 RNA polymerase produced after induction specifically transcribes the coding sequence of the protein of interest that is inserted into the expression plasmid under the control of the T7 promoter 6,7. Moreover, access to the plasmidic T7 promoter can be regulated by the lacI repressor when the T7 promoter is fused with the lac operator (T7lac promoter) 8. Several strategies have been developed over the past decades to improve the induction of expression in E. coli. IPTG is currently the most efficient method to induce promoter expression. However, this technique has the following limitations: (i) it requires cell culture monitoring to ensure that IPTG is added at the optimal cell density. Indeed, the induction point varies greatly from one recombinant protein to another, which makes the process difficult to automate, especially when several proteins are expressed in parallel (e.g., for a screen); (ii) it presents technical issues for small volumes; (iii) it is not compatible with industrial scale-up; (iv) it presents toxicity limitations (especially for human therapeutic protein production) 9 ; and (v) it is not cost-effective. The T7 system results in low recombinant protein expression during bacterial growth prior to induction. This phenomenon, which is commonly known as leaking, limits cell growth in cases of toxic recombinant protein production. Different approaches were designed to minimize or prevent this so-called leaking. Grossman et al. reported that the addition of 1% glucose to the medium led to the repression of the lac operon 10. Another strategy consisted of inserting a plasmid encoding the T7 phage lysozyme into its namesake BL21(DE3)pLysS strain. The T7 lysozyme binds to the T7 RNA polymerase and inhibits transcription initiation, thereby lowering the