Prospects for The Development of High-throughput Recombinant Protein Expression in Escherichia coli

The ease of genetic manipulation, low cost, rapid growth and the number of previous studies have made E. coli one of the most widely used microbial species for the production of recombinant proteins. In this post-genomic era, rapid expression and purification of large numbers of proteins for academic and commercial purposes in a high-throughput manner are still facing challenges. At present, several state-of-the-art methods are suitable for the cloning, expression and purification of a large number of molecules, and the latest developments related to soluble protein expression, mRNA folding, fusion tags, post-translational modifications and membrane protein production are discussed.

High-throughput research can be defined as research that allows the measurement results of thousands of biomolecules to be obtained at the same time, thus making large-scale repetitions possible. In the post-genomic era, the use of high-throughput technologies for measuring DNA, RNA, proteins, lipids, and metabolites has increased dramatically. These technologies have been successfully applied to answer various biological questions related to cancer biology.

Protein expression and purification play a central role in biochemistry. Recombinant proteins can be expressed using prokaryotic systems (E. coli and Bacillus subtilis), eukaryotic systems (yeast, insect cells and mammalian cells) or in vitro systems. The E. coli system is the host of choice for preliminary screening of recombinant protein expression, because these cells can be easily manipulated, cultivated inexpensively, and grow rapidly. In recent years, many new strains, vectors, and tags have been developed to overcome the limitations of the system, including codon bias, inclusion body formation, toxicity, protein inactivation, mRNA instability, and lack of post-translational modifications.

It has been extensively studied in the E. coli expression system, but it is labor-intensive and time-consuming to use this system for protein expression and purification. Therefore, parallel, high-throughput methods must be used for protein expression and purification, which has always been the bottleneck of protein function, structure and application research in the post-genomic era. High-throughput protein production methods are widely used, and recombinant proteins in the form of inclusion bodies are even expressed and purified in a parallel manner.

An effective promoter for expressing heterologous proteins in E. coli has four key features: First, the promoter is strong enough to allow the accumulation of recombinant protein to be greater than or equal to 10-30% of the total cell protein; second, it shows The minimum basic transcriptional activity is obtained, so unnecessary transcription is avoided before induction; third, the promoter can be induced simply and cheaply; fourth, the promoter activity can be precisely adjusted.

The selection of strains for expressing recombinant proteins also plays an important role in protein expression, solubility and yield. A few E. coli strains such as BL21 and its derivatives are widely used. Different E. coli strains promote the expression of proteins containing disulfide bonds or proteins encoded by genes that contain rare codons and proteins that are toxic to E. coli. In addition, co-expression with some genes increased the expression of post-translationally modified proteins. So far, several E. coli strains that can significantly increase membrane protein production have been designed

Successful recombinant protein expression and purification are often essential for basic research as well as for biotechnology and commercial applications. High-throughput protein expression and purification in Escherichia coli have begun to revolutionize the research methods in various research fields. Experiments that are usually performed manually are processing one protein at a time in a few weeks, and now hundreds of proteins can be performed in just one week. However, limitations still exist, and further improvements are possible.

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