Preparation method of microorganism ribosome and application of microorganism ribosome to preparing recombinant protein

Abstract

The invention discloses a preparation method of microorganism ribosome and application of the microorganism ribosome to preparing recombinant protein. The method for preparing ribosome is provided. The method includes the steps that (a), ribosome crude extracts are prepared; the ribosome crude extracts are prepared in the following steps that (a1) microorganisms are taken and are subjected to high-pressure cell smashing, and then liquid supernatant is collected in a centrifugal mode; high pressure is higher than 500 Pa; (a2) the liquid supernatant obtained in the (a1) step is taken, impurity protein is removed in an ammonium sulfate precipitation mode, and liquid supernatant is collected in a centrifugal mode; the concentration of ammonium sulfate used for removing the impurity protein in the ammonium sulfate precipitation mode is 1.5 M. The purity of the prepared ribosome coming from thermophilus is high, the structure is complete, the ribosome can be used for a thermophile in-vitro cell-free translation system and used for in-vitro expression of various kinds of thermophilic protein and research in the aspect of heat stability evolution of medium-temperature protein.

Description

technical field [0001] The invention relates to a preparation method of microbial ribosome and its application in preparation of recombinant protein. Background technique [0002] Protein is the basic functional unit of life, and the analysis of its mechanism of action is the key to understanding the function and mechanism of biological systems. The protein structure revealed by X-ray crystallography, cryo-electron microscopy and nuclear magnetic resonance (NMR) provides intuitive evidence for the analysis of the protein's mechanism of action, and the key step in this process is the preparation of recombinant proteins, and the cell-free protein translation system is Protein preparation provides effective protection. [0003] Compared with the intracellular expression system, the cell-free protein translation system can express proteins that are toxic to cells, and the protein product can be isotopically labeled or unnatural amino acids can be added to the product to facilit...

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Problems solved by technology

[0004] The cell-free translation system has been widely used in basic research and applied research in the past decade due to its following important advantages: 1) The cell-free translation system is easy to operate and can purposefully add amino acids with specific markers in proteins, such as There are radioactive elements, etc., which are convenient for the determination of the NMR structure or X-ray crystal structure of the target protein; 2) In the post-genomic era, the high-throughput protein expression, synthesis and purification platform is of great significance, and the cell-free in vitro expression system can be used for large-scale Large-scale protein synthesis provides a powerful tool for proteomics research; 3) The existing cost of extracting pharmaceutical proteins from tissues is expensive, which greatly hinders the extensive research and application of pharmaceutical proteins, while the cell-free translation system can Greatly reduce the preparation cost of pharmaceutical proteins, so that the wide application of pharmaceutical proteins can be popularized; 4) The cell-free translation system can also be used in the research of protein engineering or enzyme engineering, especially, directed evolution is one of the important means to improve the functional properties of proteins 1. Methods such as mRNA display and ribosome display based on the cell-free translation system have further broadened the research approach of protein evolution; 5) The cell-free translation system is conducive to the synthesis and expression of membrane proteins, which are the key components of many pharmaceutical molecules. However, due to the special structure of membrane proteins, it is difficult to overexpress in cells, and its high expression is often an important limiting factor restricting the study of the structure and function of membrane proteins; 6) In addition, cell-free translation systems also help virus-like particles According to research, virus-like particles are complexes assembled spontaneously by one or more proteins, which are very similar in structure to viruses and can replace viruses to activate the immune system. Since they do not contain genetic material, they can be used as safe vaccines for clinical use. The production of virus-like particles in vivo is limited by the influence of the cell environment. Firstly, the amount of preparation is limited. Secondly, the expression in vivo is easily contaminated by other in vivo factors. Clinical application has a great risk. However, the cell-free translation system can produce a large number of clinically safe virus-like particles. particles

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