Advantages of prokaryotic expression system

  • We all know that there are many ways of protein Expression, such as mammalian cell Expression system, insect cell Expression system, This time, I would like to introduce the expression of bacillus .

    Bacillus expression is a gram-positive bacterium widely used in industrial production. Different from Yeast Expression Systems, bacillus subtilis has no outer membrane, and the secreted protein can be directly released into the culture medium. So as a prokaryotic expression system with great potential, in what ways is it strong?

    1. Safe and reliable

    The safety of bacillus subtilis is food-grade and is included in the FDA's GRAS bacteria. The European food safety authority believes that bacillus subtilis can be used for food fermentation. It can be found in many common food processes. Compared with escherichia coli, bacillus subtilis has simple cell wall composition, only containing peptidoglycan and phosphoteichoic acid, and there is no mixture of heat-source lipopolysaccharide (endotoxin) and other substances in the secreted protein products similar to the cell wall components of gram-negative bacteria. The expression system can be used to purify the expression of medicinal protein without removing endotoxin.

    2. High yield and high efficiency

    Both e. coli and bacillus subtilis express and accumulate large amounts of target proteins in a short time. But e. coli in the subsequent need to collect the bacteria in the fermentation process to break the cell processing, and bacillus subtilis thanks to its itself have a set of efficient secretion signal peptide and molecular chaperone systems, simply deal with fermented supernatant can get relatively pure protein, and in most cases has a natural expression of protein conformation and biological activity.

    Proteomics studies have shown that there are at least four protein secretory pathways in bacillus subtilis, and Sec secretory pathway is the main secretory pathway. In addition, bacillus subtilis has a good fermentation technology, most of the commercial proteolytic enzymes and amylases are obtained from its fermentation.

    Although bacillus subtilis has unlimited potential to express exogenous proteins, it also has its shortcomings, such as degradation of the expression products by its own extracellular protease, relatively difficult genetic operation, and sometimes the exogenous proteins cannot be effectively expressed, etc.. However, we can successfully avoid the above problems and effectively express the foreign target protein in the expression system of bacillus subtilis, which mainly benefits from the following three advantages:

    (1) Rich expression of the host

    Because bacillus subtilis has no obvious codon preference, it is not necessary to optimize the DNA sequence when expressing exogenous proteins. Protein expression may vary in different hosts. Among the expression hosts of various bacillus subtilis,1012 wild type host bacteria is the most commonly used, which can be used as the universal bacteria for intracellular and extracellular expression. Marburg, with clear genetic background, is the standard strain for the study of bacillus subtilis. AS1 is often used to express exogenous proteins as a backup strain of intracellular expression. However, the extracellular protease-deficient strain WB800N can effectively solve the degradation of protein expression products caused by the abundant proteases in bacillus subtilis.

    (2). Expression of the vector

    Most bacillus subtilis does not contain endogenous plasmids and does not generally recognize promoters in e. coli. The original bacillus subtilis vector originated from staphylococcus aureus, which is unstable and easy to lose. The plasmid vector commonly used now is the shuttle vector composed of bacillus subtilis crypto plasmid and escherichia coli plasmid, which can complete genetic operations such as connecting and cloning of gene fragments in escherichia coli, and then transfer to bacillus subtilis for exogenous gene expression.

    (3). Effective means of transformation

    In the early stage, the most common transformation method of bacillus subtilis was Spizizen transformation (conjugation transformation), but few strains could spontaneously form the receptive state in the growth cycle of bacillus subtilis, and the retention time of the receptive state was short. Electrochemical conversion method is simple to operate and has a relatively high conversion rate. By optimizing the conditions of electrochemical conversion and applying it to the transformation of bacillus subtilis, the conversion time can be effectively shortened and the positive conversion rate can reach more than 90%. Compared with escherichia coli, the expression system of bacillus subtilis is not very perfect, I believe that with the deepening of its research, it will be in the future development of great brilliance. After all, the advantages of short cycle, low cost and high expression level of bacillus subtilis expression system over eukaryotic expression system are still attractive. Of course, we also look forward to more new expression systems that can serve scientific research and benefit mankind.