T7-lac Promoter Vectors Derepression Caused by Plant-Derived Growth Media Can Lead to Serious Expression Problems: A Systematic Evaluation.

Background The widespread usage of protein expression systems in Escherichia coli ( E. coli ) is a workhorse of molecular biology research and practical applications in biotechnology industry, including pharmaceutical drugs production. Various factors can highly affect successful clones construction and their stable maintenance as well as obtained biosynthesis levels. These include correct selection of recombinant hosts, expression systems, promoters regulation, repression level at uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics and chaperons presence/absence, among others. However, the optimization of the growth media compositions is often overlooked. We systematically evaluate this factor, which can have dramatic effect on biosynthesis of recombinant proteins, especially those, which are toxic to a recombinant host. Results Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed ORFs with wide spectrum of toxicity to the recombinant E. coli : ( i ) gfpuv (nontoxic); ( ii ) tp84_28 – coding thermophilic endolysin (moderately toxic) and ( iii ) tthHB27IRM – coding for thermophilic restriction endonuclease-methyltransferase (REase-MTase) (very toxic). The use of plant-derived peptones (soy peptone and wheat extract) in culture media causes leakage of the T7- lac expression system. 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 cultures, clones stability and biosynthesis levels. Conclusions The use of plant-derived peptones in culture media when using the 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 toxic to a recombinant host, this can result in the expression vector and/or cloned gene mutations, deletions, host’s death or highly decreased expression levels. This phenomenon is caused by certain saccharides content in plant peptones, some of which (galactosides) may act as T7- lac promoter inducers by theirinteraction with Lac repressors. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or use them with caution and to pilot-scale evaluate the derepression effect on the case-by-case basis.