Translational machinery engineering for soluble expression of recombinant protein in Escherichia coli

Abstract

The recombinant protein production in Escherichia coli has many advantages such as low culture cost, simple scale up process, well-known genetic information, and rapid accumulation of biomass. However, it is limited due to protein misfolding that leads to produce non-active protein. Although many attempts have been made for increasing solubility of recombinant protein including culturing at low temperature, co-expression of molecular chaperone, and directed evolution, it is still remained as tricky problem until now.

Protein folding is occurred co-translationally and its structure is determined by folding intermediate structure during translation process. Protein folding pathway is a trace of folding intermediates synthesized in consecutive order with growing polypeptide chain. Native like structure of folding intermediates introduces to active form, whereas if it is fail to form native like structure, it will be misfolded. When recombinant protein is expressed in E. coli, it would be misfolded by difference of folding pathway. Therefore it is important to control protein folding pathway to increase solubility of recombinant protein.

The structure of protein folding intermediate is affected by translation elongation rate which determines protein folding reaction time or number of amino acids participated in folding events. Therefore, control of translation elongation rate would be key factor for driving protein folding pathway toward producing native structure.

Here, a novel approach was developed for functional expression of recombinant protein using elongation factor G (EF-G) to control translation elongation rate. EF-G helps translocation of ribosome during translation elongation process and modified catalytic activity of EF-G causes change of elongation rate.

In chapter 2, to modulate elongation rate for controlling folding pathway, elongation factor G was modified in its primary structure to perturb catalytic activity. In addition, genetic circuit was constructed to switch between growth optimal elongation rate by wild type EF-G and production optimal elongation rate by EF-G mutant. For constructing genetic circuit, cI repressor, transcription factor, and inducible promoters were used to switch elongation rate by IPTG induction.

In chapter 3, the developed system was applied to increase solubility of wild type GFP as model system. Wild type GFP was useful as model system to validate soluble expression system because it is expressed as inclusion body in E. coli and its activity can be easily measured by detecting fluoresce. To generate various elongation rates, EF-G mutant library was constructed through PCR based mutagenesis and library was transformed into the engineered strain with expressing wild type GFP. EF-G mutant library with expressing wild type GFP was analyzed using fluorescent activated-cell sorter (FACS) and isolated to mutant with high fluorescence fraction from mutant library. Among isolated library, mutant 91 was improved as fluorescence comparing with control strain and its solubility was increased.

In chapter 4, reporting system with antibiotic marker was developed in order to efficient screening. This system is able to convert to selective phenotype such as antibiotic resistance from solubility which cannot be easily screened. To validate screening performance of this system, GFP variants fused ampicillin resistance gene at C terminus was applied. Three GFP variants, wild type GFP, eGFP, and sGFP, were shown as different ampicillin resistance due to their different solubility. It indicated that each variant could be discriminated by ampicillin concentration and screening system would be good to screen strain with expressing high soluble recombinant protein after protein engineering.

Lastly, in chapter 5, developed expression system was applied to express human granulocyte colony stimulate factor (hG-CSF). hG-CSF is a major therapeutic protein and is expressed as inclusion body in E. coli resulting low production yield after purification. To increase soluble fraction of hG-CSF, hG-CSF was expressed in EF-G mutant library and eGFP reporter was fused at C terminus of hG-CSF to easily screening from mutant library. After generating mutant library, it was screened by FACS and isolated with high fluorescence fraction. Consequently, fluorescence of mutant 4 was 3 times higher than that of control strain with expressing wild type EF-G after Isopropyl-β-D-thiogalactopyranoside (IPTG) induction. In addition, its solubility was increase about 10 percent whereas soluble fraction of control strain was not detected.

Collectively, in this study, for solving the protein misfolding problem, elongation rate was modified to change protein folding pathway because structure of protein folding intermediate is related with elongation rate and guiding protein folding structure toward forming native structure can be achieved by modulating protein synthesis rate. Modulation of translation elongation rate was achieved through elongation factor G engineering and synthetic genetic circuit was constructed to switch elongation rate to heterologous expression optimal from growth optimal in order to obtain maximum production yield. Developed expression system is useful for soluble expression or recombinant protein and it is proved by expressing wild type GFP and hG-CSF.

In the future, the developed soluble expression system can expand strategy for production of recombinant proteins with previous studies such as culturing at low temperature, co-expression of molecular chaperone, and directed evolution and also it can be applied for the soluble expression of various recombinant proteins having misfolding problem such as therapeutic protein, industrial enzymes, or heterologous expression for metabolic engineering in E. coli.