Development of novel systems for soluble expression of recombinant protein in Escherichia coli

Abstract

Recombinant protein expression technology has made it possible to economically produce various useful proteins. Many diseases, including diabetes and growth disorders, are associated with the absence or dysfunction of proteins. To date, gene therapy has not been widely used, so administering the protein is a reasonable treatment. However, in a naturally produced host, the target protein is present in a very small amount and contamination may occur in the process of obtaining, so the process of obtaining the protein becomes very inefficient and dangerous. Therefore, it is ideal to obtain the protein by recombinant protein expression technology. Host systems for producing recombinant proteins range from E. coli to mammalian cells. Among them, E. coli is a host system widely used in the production of recombinant proteins due to its advantages such as low cell culture cost, protein yield, ease of scale-up, and ease of cell manipulation. However, this system has a major problem affecting the biological activity of proteins due to poor protein folding. Therefore, the problem of folding the recombinant protein in E. coli is an issue that must be solved. Various attempts have been made to solve the folding problem when using E. coli as an expression host. First, it was confirmed that the folding problem was improved in various proteins when cultured at a low temperature. Second, a variety of E. coli strains engineered to overcome the problem of poor folding are commercially available. For example, Rosetta and Codon-Plus were designed to overcome the codon bias problem by further expressing the tRNA expression gene. In addition to this, many strategies such as altering culture parameters, co-expression of chaperones, altering gene sequences, or fusing solubility partners have been reported to work well for some proteins, but it is still impossible to obtain soluble proteins for most cases. However, these strategies have different results depending on the target protein and are insufficient to be used as a universal tools for improving protein folding. Therefore, it is necessary to develop tools that can be used universally for the expression of soluble protein in E. coli. The first system to be developed in this study is a folding reporter system using essential proteins. Recently, several approaches have been developed that can directly screen for improved protein folding in vivo. The most widely used folding reporter is green fluorescent protein. It was reported that the solubility of the protein was closely related when expressed by linking the recombinant protein with the green fluorescent protein. The advantage of the fluorescent protein reporter is that it enables high-throughput screening based on 'Fluorescence-Activated Cell Sorting (FACS)'. On the other hand, selective phenotype is another powerful method, and antibiotic-resistant proteins have also been widely used as folding reporters. The advantage of the antibiotic-resistant protein reporter is that it enables high-throughput screening based on 'Selection', which enables strains with high solubility to selectively survive when treated with antibiotics. However, these folding reporter systems (green fluorescent protein and antibiotic resistant protein) are powerful screening methods, but also have the disadvantage that false positives are observed very often. Therefore, in this study, a new system was designed to convert protein solubility into selective phenotypic cell growth. Since essential genes are an integral part of cell growth, genetic circuits have been devised to separate the growth and selection phases. Essential genes for this were selected and tested by several criteria. Next, a strain capable of converting the essential gene was constructed, and it was confirmed whether the target protein and the fusion protein composed of the essential protein were expressed to change growth according to the solubility of the target protein. Finally, a mutant library of the target protein will be constructed and applied to the protein folding reporter system. The second system to be developed in this study is a protein translation rate control system. The protein folding structure is determined at the protein translation stage, and the initiation and elongation stages play an important role. First, the protein elongation process is a process of linking amino acids to growing polypeptides. Since the formation of the protein folding structure in E. coli occurs simultaneously with protein translation, the formation of the folding structure may be very different depending on the elongation rate. Several studies have shown that protein elongation has a significant effect on protein folding structures. Next, the protein initiation process can also affect the protein solubility. The rate of translation initiation affects the mRNA ribosome occupancy and ultimately the mRNA secondary structure. The secondary structure of mRNA affects elongation because ribosomes have to unpack and translate. In addition, the rate of protein initiation affects the amount of protein synthesis, which may accelerate the formation of an 'inclusion body' when it deviates from the capacity of chaperones in cells. Therefore, in this study, a system was designed to increase protein solubility by controlling the protein translation rate at the initiation and elongation stages. The EF-G mutant library for elongation rate regulation was constructed by error-prone PCR-based random mutagenesis and site-saturation mutagenesis. The mutant library was used to evaluate whether the elongation can be controlled by the EF-G mutation. Next, several target proteins were selected and how the protein solubility in the EF-G mutants was changed. Afterwards, it is planned to build a library with various elongation rates and various initiation rates and screen for variants with increased solubility. Taken together, in this study, two systems were developed to improve the folding of recombinant proteins and increase their solubility. First, a protein folding reporter system was developed using essential proteins to effectively select various mutant libraries of proteins. Second, by developing a system that regulates the rate of protein translation, a strain having a translation rate that maximizes the solubility of the target protein can be effectively selected. These methods can be used as tools to solve the folding problem that is the most problematic when expressing recombinant proteins in E. coli, and to economically produce useful proteins.