(127c) Hyper Extracellular Production of Single-Chain Variable Fragments (scFvs) Using Recombinant E. coli By Fed-Batch Culture Based on Do-Stat

Recombinant protein production is one of the fundamental techniques in the biotechnology field. Though there are many hosts to produce recombinant proteins including, Escherichia coli, yeast and animal cell, the E. coli is still primary choice for recombinant protein production because the expression systems are well established and they are easily cultivated at low cost. However, there are some drawbacks in the protein production by E. coli such as the formation of insoluble inclusion bodies, little extracellular production and no glycosylation mechanism of proteins. Among them, the aggregation of proteins into insoluble form called inclusion body seems to be the serious problem in the recombinant protein production using E. coli because the inclusion body of proteins requires the complicated purification steps including cell disruption and refolding steps to obtain the correctly folded proteins. Therefore, it is very beneficial if we can obtain the soluble target proteins by the E. coli expression system.

At the moment, several strategies are available to improve the solubility of the expressed proteins in E. coli. To avoid forming inclusion bodies, it is known that the control of protein synthesis rate is effective. To control the protein synthesis rate, some approaches including the use of low copy number plasmid, the growth rate control by glucoses feeding and/or temperature lowering are used. The secretion of target protein into periplasm is also effective to achieve the smooth formation of disulfide bonds. Furthermore, To achieve the cost-effective production of proteins, it is advantageous to employ the high density culture of E. coli by a fed-batch culture.

In this study, we investigated the extracellular production of active target proteins by E. coli based on the following two approaches. First, to improve the solubility of protein, we examined the introduction of periplasmic secretion signal which promotes protein secretion into periplasm. Then, to achieve high density culture of E. coli, we applied a fed-batch culture with DO stat strategy. As model proteins, single chain variable fragments (scFvs) were employed. A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (V_H) and light chains (V_L) of immunoglobulins linked with a short linker peptide. Whole antibody needs to be produced by the expensive animal cell culture, but scFv can be produced by E.coli at low cost. Therefore, many researchers are working on the medical and diagnostic applications of scFvs instead of whole antibodies. To our knowledge, the scFv production by E. coli in previous research is 0.5-3.5 g/L as inclusion bodies and 5.0-100mg/L as extracellular soluble proteins.

MATERIALS AND METHODS

The pET expression system with strong T7 promoter was employed for the expression of proteins in this study. Several anti-CRP scFv and anti-human IgG scFvs were used as model proteins. Target genes are cloned between NcoI and NotI site in the pET22(+) plasmid and transformed into the host Rosseta2(DE3). It is said that the copy number of plasmid in pET system is medium (10-20 copies/cell). A periplasmic secretion signal, pelB leader was involved in the pET22(+) plasmid at the downstream of Lac operon. Because a histidine tag is added at the downstream of NotI for purification, the cloned scFv was expressed as a protein fused with the pelB leader to the N-terminus and with the his-tag to the C-terminus.

To achieve the high density culture of E. coli, we performed fed-batch cultures using a computer-controlled jar-fermentor with DO stat.Fed-batch culture is a traditional operation technique of bioreactor where substrate such as glucose is fed to bioreactor until the end of cultivation without withdrawing culture broth. By controlling substrate feeding rate properly, a high density culture becomes possible, so a fed-batch culture is widely employed in the industrial operation of bioreactors. During the fed-batch operation, if too much glucose is fed into a bioreactor, dissolved oxygen (DO) in a bioreactor will decrease. On the other hand, if glucose supply is too less, DO will quickly increase. DO-stat strategy used in this study is to feed glucose so as to maintain DO in a bioreactor constant. DO-stat strategy is simple but it can realize a high density culture of active E. coli. Because there is a delay of DO change in response to glucose feeding, DO concentrations become unstable when applying a simple ON-OFF control. To improve the accuracy of DO control, we employed a PID control to determine the feeding rate.

RESULTS and DISCUSSION

First, we checked scFvs production using five recombinant strains harboring scFv genes by flask cultures. Cell growth was successful for all experiments and 1.3-2.9 g/L of scFvs were produced mainly as inclusion bodies. Small amount of extracellular scFv secretion was observed for the strains introduced pelB leader and their solubility was 2.6-5.2%. From the results of SDS-PAGE, it was found that many kinds of intracellular proteins including scFv were leaked into culture broth by the introduction of pelB leader. From the results of western blotting, scFv were secreted into culture broth only when the pelB leader was introduced. These results suggested that the E. coli with the PelB leader sequence had a potential to realize the extracellular production of scFv under a long-term operation such as a fed-batch culture.

The operation of the fed- batch culture got started by a batch culture using the complex medium containing 20g/L glucose. If most of glucose in a fermentor is consumed, the fed-batch operation was started at the agitation rate of 800rpm and DO was set at 40% of saturation. Then, the OD600 becomes 60, IPTG was added for the induction and the fed-batch operation was continued about 100 hours. During fed-batch operation, DO in a fermentor was successfully maintained at around 40% as set. The cell growth was very smooth and OD reached 130 at 55 hours. The insoluble scFv was accumulated first, however, after 30hours, soluble scFv was gradually accumulated in a culture broth and continued till the end of cultivation. The final scFv in supernatant reached 4.8 g/L. This is extremely high compared with previous research because it has been recognized that the extracellular protein production by E. coli is basically impossible. Binding affinity of the secreted scFv was confirmed by SPR sensor.

Then, we conducted the fed-batch experiment using different scFv strain, anti-human IgG scFv. DO in a fermentor was also maintained at around 40% as set. Time courses of fed-batch culture was almost similar to those of previous experiments and soluble scFv in the supernatant was gradually accumulated in a culture broth and the final scFv in supernatant reached 3.1 g/L.

By applying the fed-batch operation for 100h, it was found that 3.1 to 4.8 g/L of the soluble scFv was secreted into culture broth. The extracellular scFv concentrations observed in these experiments were extremely high compared with previous research. The solubility of the expressed proteins was approximately 52-63%. The possible mechanism for scFv secretion into culture broth is considered as follows at the moment. The expressed scFv with the pelB sequence was directed to the periplasm by the pelB leader and the pelB sequence is removed by a signal peptidase in the periplasm. The concentration of scFV in the periplasm continued as the fed-batch culture progressed. On the other hand, as previously mentioned, the pelB leader influences the membrane permeability and promotes the secretion of intracellular proteins through the outer membrane. By applying fed-batch operation under these circumstances, the protein expression will continue for a long time, resulting in the hyper extracellular production of scFv. Since it is very easy to purify the soluble scFv in supernatant, these results will greatly contribute to decrease the purification cost in the recombinant protein production.