The effect of selected peptones on the T7-lac expression system leakage
While conducting various cultures of recombinant E. coli, aimed at expressing cloned genes in the Tabor-Studier system (pET-series), we observed that despite employing identical growth conditions, cultures sometimes behaved differently. This concerned both the cultures grown under non-inducing conditions as well as upon induction to the overproduce recombinant proteins. After analyzing the series of control experiments (not shown), the only factor that differentiates the erratically behaving E. coli cultures, carrying the same genetic constructs, was the use of soy peptone or peptone tryptone during the preparation of the culture media. To further investigate this phenomenon, a number of E. coli BL21(DE3) cultures carrying one of the 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 (nontoxic); (ii) tp84_28 – coding thermophilic endolysin (moderately toxic) and (iii) tthHB27IRM – coding for thermophilic REase-MTase (very toxic). The GFPuv protein, which is very well tolerated by E. coli, has an additional advantage in our expression evaluation that can be assayed in whole cells, not only by SDS-PAGE electrophoretic analysis, but also by exposing the bacteria to UV light. This feature makes it very well suited for this study. The second protein selected for analysis was the TP-84 bacteriophage endolysin, which executes a moderate toxic effect on host cells – its pET vector-based expression clone is not problematic to maintain in uninduced state, while upon induction the recombinant E. coli the cells become fragile and prone to spontaneous lysis, apparently due to a penetration of small amounts of the protein through the cytoplasmatic membrane and a degradation of the peptidoglycan layer. As the third protein in our study, the recombinant TthHB27I REase-MTase was selected, originating from Thermus thermophilus HB27I. Due to its unbalanced REase versus MTase activities in the recombinant E. coli host cells, it is very toxic, frequently causing the recombinant E. coli cells lysis or mutants accumulation during induced expression. Sometimes those problems were evident even in the uninduced state [29, 34]. Thus, to obtain the adequate biosynthesis level of the TthHB27I protein with biological activity, is it essential to maintain a mutant-free and not prematurely dying E. coli population carrying the pET vector-based expression construct, by exercising strict control of the T7-lac promoter. For overproduction of test proteins, E. coli BL21(DE3) cells were transformed with the plasmids: pET21d(+)-gfpuv, pET21d(+)-tthHB27IRM and pET21d(+)-tp84_28, then plated on LA medium in 9 variants described below and incubated overnight at 30°C. At higher incubation temperatures, there are no or very few transformants carrying
the tthHB27IRM gene [29]. In the case of the endolysin clone both 30°C and 37°C incubations resulted in obtaining correct colonies, nevertheless the 30°C incubation was used as a precaution. For GFPuv, no toxic effect was observed, but overproduction of this protein at lower temperatures is advisable as better suited for correct GFP protein folding and solubility [35]. The media variants contained different types of peptones or no peptone in the case of the control culture. No IPTG or other gene expression inducer acting on the T7-lac promoter was added. After overnight incubation, the bacterial colonies were observed on plates with all media (for all expression plasmids). The plates with bacteria carrying the plasmid pET21d(+)-gfpuv were exposed to UV using a transilluminator (Fig. 1). As clearly seen on Fig. 1B, the bacteria that grew on the LA medium made with soy peptone showed a strong green fluorescence, which indicates the presence of GFPuv protein overexpression. The green fluorescence was also observed in the case of the wheat extract substrate (Fig. 1C). However, its level was significantly lower. The fluorescence was not observed for the bacteria that grew on the other media tested (Fig. 1A, D, E, F, G, H, I), indicating no or very low T7-lac promoter leakage. Nowadays, when expressing cloned genes, constitutive promoters are being rarely used. To the contrary, expression systems with a strict process control are typically used. However, due to the imperfections associated with each expression system, a number of methods have been developed to increase their tightness. In the case of protein overproduction in the Tabor-Studier system, these include: (i) co-expression of the gene encoding the protein of interest with the additional copy of the gene encoding the LacI repressor; (ii) the gene coding for T7 lysozyme, which is an inhibitor of T7 RNA polymerase; (iii) the introduction of the lacO operator behind the T7 promoter sequence, forming a fusion T7-lac promoter [36]. Also, decreasing the temperature down to even below 20°C for certain engineered E. coli strains helps to control a recombinant protein deleterious activity. Even a small level of background expression can have deplorable effects in the case of overproduction of toxic proteins in the form of slower growth rate of the culture, decreased protein biosynthesis level, mutants accumulation, plasmid loss, low culture cell density or cell death [37, 38]. To illustrate this effect, E. coli BL21(DE3) cultures carrying the 3 test plasmids in liquid media of different composition (analogously to cultures on solid media) were performed. SDS-PAGE analysis of the cellular proteins profile at individual stages of the cultures showed that the increase in GFPuv biosynthesis (Fig. 2) can be observed in uninduced cultures carried out in a medium containing either soy peptone or wheat extract. The amount of GFPuv protein produced in both cases is very different – soy peptone usage resulted in a massive expression of GFPuv, visible as a dominating band (26.8 kDa, marked as red arrows) on SDS-PAGE (Fig. 2B, C). However, in other peptones’ cases, no proteins corresponding to the control protein size (purified GFPuv preparation) were observed (Fig. 2A, D, E, F, G, H, I). This result coincides very well with the results of the cultures carried out on solid media. However, more information was obtained concerning the effect of uncontrolled protein overproduction on the culture. An effect somewhat similar to the GFPuv was observed during the SDS-PAGE analysis of the profile of cellular proteins at the individual stages of the culture producing endolysin (44.2 kDa, red arrows) clone, although the protein biosynthesis level was much lower (Fig. 3). The situation is different in the case of the cultures carrying the pET21d(+)-tthHB27IRM plasmid., where the appearance of a protein was observed of the size corresponding to the control protein (purified TthHB27I preparation, 127.7 kDa) in the culture grown in the presence of soy peptone (Fig. 4). Analyzing the growth curves of individual cultures (Fig. 5), several observations can be made. The control cultures, in all cases, exhibited weaker growth, which is was expected due to the limited source of nutrients (yeast extract only). The cultures grown in the media containing various peptones of animal origin showed similar growth kinetics. However, the most striking is the comparison of the course of all the cultures carried out in the media containing plant derived peptones. For E. coli BL21(DE3) [pET21d(+)-tthHB27IRM] cultures (Fig. 5B), a drastic decrease in cell density and spontaneous cells lysis (between 5 and 7 hours of cultivation) was observed in the media containing soy peptone, typical for T7-lac promoter-induced cultures overproducing proteins toxic to E. coli host cells. This result coincides with a large increase in TthHB27I protein in cells, as seen on the SDS-PAGE gel (Fig. 4B, samples for points T5 and T6). The optical density of the E. coli BL21(DE3) [pET21d(+)-tthHB27IRM] and E. coli BL21(DE3) [pET21d(+)-tp84_28] cultures carried out in the presence of wheat extract was the lowest among those grown in a medium with complete composition. To the contrary, E. coli BL21(DE3) [pET21d(+)-gfpuv], producing nontoxic GFPuv, showed the fastest growth on wheat extract, apparently due to the presences of large amounts of saccharides, serving as a rich energy source. In the case of E. coli BL21(DE3) [pET21d(+)-gfpuv] or E. coli BL21(DE3) [pET21d(+)-tp84_28] cultures (Fig. 5A and C), no decrease was observed in the density of the cultures grown in soy peptone media, even though, apparently, it also contained some T7-lac promoter-inducing components, as shown on Fig. 1. Moreover, these cultures achieved very high optical density values on soy peptone, which indicates the nutritional conditions favorable for the tested bacteria. However, when wheat extract was present in the culture medium, the bacteria carrying the gene encoding endolysin behaved similarly to the bacteria carrying the gene encoding TthHB27I – the growth was relatively slow but, apparently, the cells were managing to cope with the presence of small amounts of those toxic proteins. On the other hand, the bacteria carrying the gene coding for the GFPuv protein behaved just opposite in this medium (Fig. 5A). The presence of wheat extract caused a very fast increase in biomass in the E. coli BL21(DE3) [pET21d(+)-gfpuv] culture, and the OD obtained after overnight incubation was 2–4 times higher than in other cultures. This result is different than those shown by other cultures, showing that overproduction of even nontoxic protein in such massive amounts usually puts a heavy strain on cell metabolism, and this should rather adversely affect the development of culture [39, 40]. This points to the conclusion, that the metabolic stress highly depends on a given protein overexpressed, which may prove useful in the optimization of biotechnological processes.
The effect of saccharides contained in soy peptones on the Tabor-Studier system
Six of the tested peptones (gelatin peptone, casein peptone, peptone tryptone, peptone tryptose, peptone proteose and peptobak) are obtained by the enzymatic digestion of proteins of animal origin and they mainly contain amino acids, peptides and proteins and small amounts of non-inducing T7-lac promoter saccharide glycogen. On the other hand, the soy peptone is obtained from soybean meal, so it is of plant origin. The origin of this peptone has a critical impact on its composition, as plants typically contain large amounts of various carbohydrates as a storage material in addition to amino acids, peptides and proteins [41, 42]. The main soluble carbohydrates found in soybean meal (and soy peptone) are sucrose, raffinose, stachyose and verbascose. Because verbascose accounts for less than 0.5% of the dry weight of soybean meal [13], it was omitted in further studies. In order to determine, which of the above-mentioned carbohydrates may cause leakage in the T7-lac promoter expression system, further detailed evaluations have been made, concerning other carbohydrates. For the clarity of those experiments, the nontoxic protein producer - E. coli BL21(DE3) [pET21d(+)-gfpuv] - was grown in LB medium, with peptone tryptone as a nitrogen source, and supplemented with a tested sugar at the final concentration of 1 mM, when the culture reached OD600 = 0.6–0.8. The final concentration of the added sugar solution and the OD of the culture at which it was added followed standard conditions for inducing gene expression using IPTG (a synthetic gene expression inducer for the T7-lac expression system). The studies on the induction of gene expression began in the 1950s [43], and since then, it has not only been shown that β-D-galactosides have to be used for induction of promoters, controlled by lac operator, but a number of substances have been synthesized that not only do not undergo hydrolysis or metabolism, but are more effective, such as IPTG [44]. Two control cultures were carried out: negative, where no additional substance was added and positive, to which IPTG was added. Solutions of 5 carbohydrates - sucrose, raffinose, stachyose, glucose and galactose were added to the remaining cultures. The last two sugars (glucose and galactose) are the monomers that make up allolactose - a naturally occurring compound that induces gene expression from the natural lac promoters as well as from the engineered T7-lac expression systems. They are not present in soybean meal and, hence, in soy peptone, but it was important to confirm or exclude their gene expression inducing effect. The cultures were grown at 37°C for 7 hours since the moment they reached OD600 = 0.6–0.8 and samples were taken every hour for spectrophotometric and SDS-PAGE analysis. The profile of cellular proteins at the individual stages of each culture is shown in Fig. 6. In the control (negative) culture, no increase in protein corresponding to GFPuv over culturing time was observed (Fig. 6A). The cultures with the addition of glucose and sucrose gave the same result (Fig. 6C and E). An increase of GFPuv protein biosynthesis level in bacterial cells was observed for the remaining cultures (Fig. 6B, D, F, G). As expected, the largest increase in protein occurred in the control (positive) culture, where IPTG was added (Fig. 6B). The biosynthesis level of GFPuv protein in the culture with the addition of galactose (Fig. 6D) was also significant - its amount, 7 hours after addition of galactose, was comparable to the amount of the protein obtained 2 hours after induction with IPTG (Fig. 6B). In the case of addition of raffinose and stachyose, the observed increase in GFPuv protein in the cells was lower than for galactose (Fig. 6F, G). These results indicate that the carbohydrates tested, which are present in soy peptone (raffinose and stachyose) are responsible for the recombinant protein biosynthesis leakage in T7-lac-based expression systems, such as the Tabor-Studier system. The structural formulas of these saccharides are shown in Fig. 7. Both raffinose and stachyose are β-D-galactosides, which corroborates with the previous finding that various β-D-galactosides can be potential inducers of lac operator-controlled promoters. While we have evaluated a limited number of plant-derived media, it is expected that similar effects of leakage in lac operator-controlled expression systems also concern other peptones of plant origin (e.g. wheat, rice, peas, cotton or potatoes), as during their production from plant tissues, carbohydrates are not completely removed. However, due to differences in the content of individual plant components depending on their species, origin, degree of maturity and processing technology, the effect will certainly vary. Therefore, small-scale individual testing prior to scaled-up production is recommended. In the cases of very toxic recombinant proteins production in the Tabor-Studier system or other lac operator-controlled systems, it may become necessary to exclude plant-derived peptones and/or supplement them with other control circuits, such as the usage of co-expression of T7 lysozyme.