Plasmid Retention and Bacteria Growth in E Coli

Plasmid Retention and Bacteria Growth in E Coli The increasing interest by the industry in recombinant protein production has caused an intensive study in this area during the last years. However, it is well known that there are a number of issues associated with the high expression of a recombinant protein. E. coli is one of the most used organisms for this purpose. In this organism, the most common and challenging problem is the formation of inclusion bodies. Probably, an incorrect folding process provokes that the recombinant protein forms those structures. When the protein forms inclusion bodies, it is insoluble and usually useless. In order to find a proper protocol for the high production of the protein S, we have assessed the expression system which use the BL21*DE3 strain as host and the pCV05 plasmid which contains the protein S sequence fused with the His tag sequence. Growth rate, plasmid loss and recombinant expression level were assessed. We obtained a reasonable production of target protein in the insoluble fraction. Further research is needed to know whether the processing of the His tag is able to make soluble the protein from the inclusion bodies as is described by other researchers. Introduction The increasing interest by the industry in recombinant protein production (RPP), due to the number of applications it can provide, has caused an intensive study in this area in order to improve its protocols. Thus, an improvement would make possible an increment in the target protein yield and the quality production as well as to establish more efficient host and plasmid for each target protein . The most common hosts utilized in RPP are bacteria because of the capacity that they have to express almost any gen and the relative facility to modify and use their plasmids in order to produce the target protein . However, it is widely known the number of problems that these hosts have when they produce a high amount of recombinant protein. Firstly, a frequent problem is the appearance of inclusion bodies which hinder a correct recovery of the target protein produced . Secondly, the host lysis event is the other common problem in RPP in bacteria. This undesirable happening in the production of recombinant proteins can be produced for several reasons. One of them is the high level synthesis of the mRNA and the target protein . Other reasons described are the accumulation of fragments of the recombinant protein because of the proteolysis . Finally, the main cause of the problems related to RPP is the accumulation of incorrectly folded intermediates of the recombinant protein. In E. coli this fact implies general stress responses . In order to find a proper protocol for the protein S (PS) production in E. coli and know more about the RPP process, we conducted an experiment testing post-induction bacteria growth, production of target protein and plasmid retention. In this experiment the E. coli strain BL21*DE3 transformed with the plasmid pCV05 was used to express the PS fused with a C-terminal His tag (6xHis). Materials and Methods The E. coli strain BL21*DE3 transformed with the plasmid pCV05 (a derivative of pET21a plasmid) was utilized to conduct the experiment. This plasmid carries in its sequence the gene of PS fused with a C-terminal extra sequence which encodes a His tag (predicted molecular weight 60 KDa). A flask with 25 mL LB (10 g L-1 tryptone, 5 g L-1 yeast extract, 5 g L-1 NaCl) supplemented with 0.1 g L-1 carbenicillin was inoculated with a single colony of E. coli. Subsequently, this flask was incubated during 7 hours (25 °C). The next step was to inoculate the 2.5-L fermenter (ΑG CH-4103 Bottmingen ®), which contained 2 L LB supplemented with 0.5% (w/v) glucose and 0.1 g L-1 carbenicillin, with 25 mL from the flask previously inoculated and incubated during 8 hours (25 °C). Immediately before inoculate the fermenter, a sample was taken in order to use it as a blank to measure the OD650nm of the following samples. The culture was grown at 25 °C until an OD650 nm of nearly 0.6 when it was added the inducer of recombinant protein expression (IPTG). The culture was supplemented with 100  µM IPTG. After this step, the culture was grown at 25 °C during 8 hours. The pH was controlled at 7 adding 5% (v/v) HCl and 1 M NH3, and 0.03% (v/v) final concentration of silicone antifoam was added in the fermenter due to prevent foaming during the last hours of the fermentation. During that period of 8 hours, the culture was fed and samples were taken at various times. One sample was picked just before the induction (0h) and the other samples were collected at 1h, 3h, 4h, 5h, 6h and 7h after the induction in order to check the bacteria growth. Besides, a pellet of bacteria was obtained from those samples collected to check the amount of target protein by a 15% SDS-PAGE gel subsequently stained with 0.2% (w/v) Coomassie Blue using the NEB Prestained Protein Marker, Broad Range (7-175 KDa) ® as a marker. In addition, BugBuster Protein Extraction Reagent ® protocol was cond ucted for additional 0h, 3h, 4h and 8h samples due to separate the insoluble and the soluble protein fractions from the bacteria and assess them by SDS-PAGE as described above. On the other hand, 0h, 3h and 7h samples were plated onto non selective nutrient agar (NA) and later replicated onto NA supplemented with 0.1 g L-1 carbenicillin in order to assess the plasmid retention of the bacteria. Results Inducer effect in Bacteria growth It was conducted a measurement of OD650 nm of the culture during the 8h-period of growth. In order to conduct that assessment, samples were collected immediately before the induction (0h) and 1h, 3h, 4h, 5h, 6h, 7h after the induction with IPTG. After the IPTG induction the culture grew slowly until time 3h. After that time, the growth rate was increased significantly until time 5h. However, at this time the culture presented a decrease in growth until time 7h. The growth dropped specially between time 5h and 6h (Fig. 1). Plasmid retention To assess how the E. coli strain BL21*DE3 retains the pCV05 plasmid after the IPTG induction, samples from the fermenter were collected at time 0h, 3h and 7h post-induction. Serial dilutions of these samples were plated onto non selective NA and subsequently replicated onto NA supplemented with carbenicillin. The relation between the colonies grown on NA plates and the colonies grown in NA supplemented with carbenicillin give the % of plasmid retention. The plasmid retention was hardly altered during the period of 7 hours post-induction assessed. Unexpectedly, according to the results obtained, the plasmid retention 3 hours after the induction is higher than the rate at time 0h. Regarding the time 7h, the plasmid retention percentage is much lower than in time 0h and 3h (Fig. 2). Target protein yield In order to compare the target protein yield during the growth of the culture, samples were collected immediately before the induction (0h) and different times post-induction (1h, 2h, 3h, 4h and 5h). In addition, insoluble and soluble fractions for time 0h, 3h, 4h and 8h were obtained in order to know if the target protein was correctly folded (protein in soluble fraction) or incorrectly folded (insoluble fraction). These samples were assessed in a SDS-PAGE gel. According to the SDS-PAGE profiles, although the PS::His band should be around 60 KDa, in this experiment the target band seems to be below the expected weight (Fig. 3). Regarding the total protein assessment, this band shows a gradual increase in its intensity from time 3h to time 5h post-induction. However, at time 0h, 1h, 2h after induction there was not significant production of the recombinant protein (Fig. 3a). As for the soluble and insoluble fraction samples, the SDS-PAGE analysis revealed a higher intensity of the target protein band in the insoluble fraction than in the soluble fraction of the times 8h, 4h and 3h. At those times, the intensity of the recombinant protein band for insoluble fraction samples is almost impossible to appreciate. The same occurs for the soluble and insoluble fraction samples at time 0h (Fig. 3b). Overall, according to the results of the SDS-PAGE profiles, there was a significant recombinant protein production since the time 3h after the induction with IPTG. On the other hand, it seems to be that there was not a proper target protein folding because the recombinant protein could be found in the insoluble fraction rather than in the soluble fraction. Discussion The main cause of an incorrect folding of the recombinant protein when a high yield is conducted is well known. The accumulation of misfolded protein intermediates causes considerable stress in the host cell . A wide range of different strategies have been conducted in order to solve this problem . It has been described that the use of IPTG-inducible T7 RNA polymerase system in the BL21 strain to produce high concentrations of recombinant protein usually implies a high level of post-induction stress . The solution proposed by some researchers is select mutants which have lower expression rates of the recombinant protein . Other researchers have opted for limiting the concentration of inducer used . Nowadays, the establishment of general protocols and host for the different target proteins is still a challenge. In this experiment, we have assessed the capacity of the E. coli strain BL21*DE3 to produce a high amount of PS fused with a C-terminal His tag as well as the bacteria growth during the process and the plasmid retention. According to the first part of the results, the bacteria growth rate changed during the 7 hours of fermentation. It is described that the high amount of recombinant protein in the cell causes stress response. This stress response implies that the growth rate of the culture turned into a negative rate (the number of cells in the culture decreases) . The data collected from other groups conducting the same experiment shows that this event has happened in 2 groups but the other 3 groups have a different growth pattern (Fig. 4). Technical issues may explain this incongruity between the different results obtained. Regarding the plasmid retention, the data obtained in this experiment suggest that because of the stress suffered by the cells when the recombinant protein levels are higher, the cells tend to have a lower rate of plasmid retention. An explanation may be that the bacteria with the plasmid suffer a higher stress due to the induction by the IPTG, and thus, they have less chance to survive than the bacteria which accidentally do not have the plasmid. Therefore, the bacteria suffer a selective pressure which results in a plasmid loss and it is more obvious after several hours of growth. Analyzing the data obtained by other colleagues, it is supported that there is a relation between the time after the induction and the plasmid retention rates (Table 1). As for the PS::His yield, the data shows that in the BL21*DE3 strain using pCV05 as a plasmid and with the conditions described before, this expression system needs 3 hours to start expressing the recombinant protein. After that time, it seems that the most part of the target protein is in the insoluble fraction. Regarding the total protein samples assessment, the relative amount of target protein produced by the cultures of the all groups was the same (Table 2). However, the recombinant protein is still in the insoluble fraction after the BugBuster Protein Extraction Reagent protocol. Probably, some of the proteins from the insoluble fraction were forming inclusion bodies . It is described that after the expression of the protein removing the His tag makes the recombinant protein more soluble and thus, it is possible to dissolve the inclusion bodies and recover a functional recombinant protein . Furthermore, the His tag allows an easier purification of the protein due to the affinit y of this polypeptide for metal ions . In order to confirm those statements for PS, it would be necessary to conduct purification and a proteolysis process of the His tag in order to assess whether the efficiency of this expression system is cost-effective and the amount of protein obtained is enough. The conclusion of these data is that this expression system for the PS yield could be a good and profitable system whether the His tag added finally allows to recover the recombinant protein from the inclusion bodies. Acknowledgements We are grateful to Dr Claire Vine, Dr Ian Cadby and Dr Jeff Cole for the excellent support given as well as the rest of the groups which conducted the same experiment because they have contributed to the experiment with very valuable data.