T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation

Daria Krefft,Piotr M Skowron,Maciej Prusinowski,Aleksandra Skokowska,Joanna Zebrowska,Paulina Maciszka

doi:10.1186/s12934-022-01740-5

Daria Krefft, Piotr M Skowron + Show 4 more

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https://doi.org/10.1186/s12934-022-01740-5

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Abstract

BackgroundThe widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium’s composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host.ResultsCommonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28—which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM—which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)—RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels.ConclusionsThe use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

Highlights

The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs
The knowledge learned during research on this operon and modifications to its components have led to a gene expression system that uses a promoter derived from the bacteriophage T7, which is not recognised by the host E. coli, but is recognised by specific T7 RNA polymerase
After analysing the series of control experiments, the only factor differentiating the erratically behaving E. coli cultures, carrying the same genetic constructs, was the use of soy peptone or tryptone peptone during the preparation of culture media. To further investigate this phenomenon, a number of E. coli BL21(DE3) cultures carrying one of genes gfpuv, tthHB27IRM or tp84_28 were performed. These test genes were selected to cover a wide range of toxicity to the recombinant E. coli: (i) gfpuv; (ii) tp84_28—which codes for thermophilic endolysin; and (iii) tthHB27IRM—which codes for thermophilic RM.TthHB27I

Summary

Introduction

The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. In addition to proteins, peptides and amino acids in soy peptone, there is a large amount of saccharides, compounds that in some expression systems can act as a gene-inducing factor. This is the case with lactose operon [15], arabinose operon [16], rhamnose operon [17] and maltose operon [18], among others. The knowledge learned during research on this operon and modifications to its components have led to a gene expression system that uses a promoter derived from the bacteriophage T7, which is not recognised by the host E. coli, but is recognised by specific T7 RNA polymerase. The T7 polymerase/promoter expression system, originally used in engineered E. coli strains, has been modified and used to overproduce proteins in other bacteria, such as Streptomyces lividans [24] or Bacillus megaterium [25], in eucaryotes (yeast) [26] and even in mammalian cells [27, 28]

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