An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli

Graphical abstract

1. Pick a single colony from the transformed LB agar plate or take 20 mL of glycerol stock and inoculate into 5 mL of LB broth containing appropriate antibiotics according to the vector construct and host cells. 2. Incubate the culture at 37 8C for overnight (14-16 h)  Note: Ethanol should be used in v/v ratio. We have optimized the optimum ethanol concentration and it was observed that 3% ethanol (v/v) gives the maximum enhancement in protein expression. In the presence of more than >5% ethanol cell growth was found to be inhibited. 6. Induce the protein expression by adding IPTG to a final concentration of 1.0 mM.

Note:
Do not add IPTG to the culture which will serve as a non-induced control. Minimal IPTG concentration should be optimized in small scale of culture before proceeding to the mass culture. The optimal growth time for TB (Terrific broth) is different from LB (Luria broth) In case of TB OD 600 should be more than 1.0-1.5 before IPTG induction. In case of auto-induction media the control should be normal LB not the auto-induction media. There is no need to observe the OD, because it does not need the IPTG induction. 11. Load the sample in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and analyze. Screening through pilot experiment is important, which will give the clear picture about the level of overexpression of the protein in the presence of 1%, 2% and 3% ethanol. This will also provide the opportunity to compare the over expression of recombinant protein in the presence and absence of ethanol. SDS-PAGE analysis will support the increase fold of expression with increased percentage of ethanol.
Note: Pilot screening can be done at low temperature ranging from 16 8C to 238C for 20-22 h after induction with IPTG, depending upon the level of expression and solubility screening of the recombinant proteins. In case of auto-induction media incubate the culture until the OD 600 reach 0.4-0.6. It is observed that some proteins give higher fold of expression in low temperature. This screening can also provide a clear idea about the level of expression of the protein, which can be helpful for further mass culture. For mass culture setup the experiment according to the same ratio of pilot experiment.

Method validation
In our laboratory, we often use the present protocol to enhance the recombinant protein expression in E. coli. SDS-PAGE results show the enhancement in recombinant protein expression in the presence of 3% ethanol in comparison to un-induced control and induced recombinant protein expression in the absence of 3% ethanol. We have taken different un-related recombinant proteins from various organisms to support our reported method, which is exceedingly helpful for enhancing the overexpression of recombinant proteins. It works with both T5 (pQE series) and T7 (pET series) type bacterial promoters ( Table 1). The method is validated in our laboratory with several recombinant proteins, including MEX67, RPB5, RPB8, RPB11 (from Saccharomyces cereviseae), amyloid-beta peptide (Ab-42 peptide) fusion protein and glutaminyl-tRNA synthetase (GlnRS, from Fasciola gigantica). MEX-67 is a 67 kDa poly(A)-RNA binding protein involved in the export of nuclear mRNA and is a component of the nuclear pore and ortholog of human TAP [1][2][3][4]. We observed that the MEX67 protein expression studied under normal IPTG induction demonstrated negligible difference in the level of proteins in comparison to the un-induced control fraction. Optimization was performed with various osmolytes and also with ethanol (Fig. 1). Best expression was found in the induced fraction treated with 3% ethanol (v/v) in the culture media [5] (Fig. 2A). RPB5 ($25 kDa), one of the small subunit of S. cerevisiae is required for yeast cell viability and play a central role in RNA transcription as it is present in all the 3 eukaryotic RNA polymerases [6]. The increase in RPB5 protein expression is shown in Fig. 2B [7]. The 16 kDa RPB8 subunit is essential for cell viability in S. cerevisiae, however, its function remains unknown [8]. The difference between RPB8 protein expression in the presence and absence of 3% ethanol is shown in Fig. 2 C. RPB11 fusion protein with GST ($40 kDa) from S. cerevisiae is a RNA polymerase II subunit that is a part of the core element with the central large cleft. RPB11 also seems to be involved transcript termination [9]. Recombinant RPB11 shows increased expression in the presence of ethanol in comparison to normal condition without ethanol as shown in Fig. 3A. Similarly,   recombinant Ab peptide fusion protein ($30 kDa) showed increased fold of expression in the presence of 3% ethanol in comparison to a condition where ethanol was not present [10] (Fig. 3B). A class I tRNA synthetase, FgGlnRS ($64 kDa) was recombinantly expressed in high quantity in the presence of ethanol as shown in Fig. 3C. RPB9 from S. cerevisiae is involved in the selection of the transcription initiation site, control of fidelity and transcription coupled DNA repair [11]. Like other recombinant proteins, RPB9 ($14 kDa) also showed increased level of expression in the presence of 3% ethanol (Fig. 3D). Method validation in a wide range of proteins from different organisms provides additional support to the success of our protocol.

Additional information
There is no report available in the literature where ethanol was used to enhance the expression of recombinant protein by many folds. Increased expression of the recombinant proteins under ethanol treatment is unique and interesting. Ethanol is an amphipathic molecule and can affect the cellular environment of the cell to a large extent by making changes in the membrane fluidity [12,13], membrane transport [12], membrane lipid composition [12,14], assembly of membrane proteins [15,16]. These changes may influence the membrane associated phenomenon, such as DNA replication, leading to the enhancement of DNA synthesis [17]. We propose that enhancement of DNA synthesis results in gene amplification that may enhance the synthesis of inducible proteins in ethanol-treated cells.
Recombinant proteins are necessary for biotechnology and pharmaceutical industries. They are also required for various R & D programme. Structural, functional or biochemical characterization of proteins requires a large amount of purified recombinant proteins. Few methods have been reported which can improve the expression of recombinant protein, but they cannot be universally utilized in comparison to our protocol. We have compared the expression fold of proteins with several osmolytes such as sorbitol, glucose, inositol and sucrose, and compared with that of ethanol. Our results showed that recombinant protein expression under the presence of ethanol gives higher fold of expression compared to osmolytes [5].
Other than enhancing expression of recombinant protein, addition of ethanol to the growth media can mimic the heat-shock response in E. coli [18][19][20] that can also help in enhancing the solubility of recombinant proteins. Few reports also suggested that the presence of ethanol results in enhancement of protein stability through stabilizing the native state of proteins [21][22][23][24][25]. Understanding the exact molecular mechanism behind the increase in recombinant protein expression using ethanol can be of enormous applications in future.

Conflict of interest
The authors declare that there are no conflicts of interest.