Method for efficiently preparing protein molecular weight standard by utilizing prokaryotic expression system

Abstract

The invention discloses a method for efficiently preparing a protein molecular weight standard by utilizing a prokaryotic expression system, which comprises the following steps of: (1) selecting a common low molecular weight standard as an initiation protein molecular weight standard A; (2) selecting a gene sequence with low codon content or without a rare codon as a template, and designing a primer to sequentially obtain different target fragments, so that the molecular weight of an encoding area after a target sequence and a pET28a vector are connected is the set molecular weight in (1), and the optimum number of isoelectric points is 4-9; (3) respectively inserting the target fragments into an expression vector pET28a-(+) according to certain enzyme cutting sites, and respectively transferring screened positive clones into a colon bacillus BL21_star(DE3) for induced expression; (4) verifying the expression forms of different proteins; and (5) respectively purifying target protein by the methods of affinitive layer purification, purification of inclusion bodies and the like, i.e. the protein molecular weight standard.

Description

technical field [0001] The invention relates to a method for efficiently preparing protein molecular weight standards by using a prokaryotic expression system, which can be applied to fields such as bioengineering and biotechnology research. Background technique [0002] Protein research is an important part of life sciences. Protein molecular weight standards can be used as an important tool for qualitative or even quantitative research on proteins. They can be used for protein electrophoresis, protein quantification, and protein immunoblotting. At present, the source of protein molecular weight standards is narrow and numerous. Most of the protein molecular weight standards are known natural proteins, the separation and purification are complicated, and the molecular weight values ​​vary greatly. Due to the complexity of natural protein modification, there are often differences in the electrophoretic mobility of the same protein from different sources, resulting in unclear ...

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Problems solved by technology

[0002] Protein research is an important part of life sciences. Protein molecular weight standards can be used as an important tool for qualitative or even quantitative research on proteins. They can be used for protein electrophoresis, protein quantification, and protein immunoblotting. At present, the source of protein molecular weight standards is narrow and numerous. Most of the protein molecular weight standards are known natural proteins, the separation and purification are complicated, and the molecular weight values ​​vary greatly. Due to the complexity of natural protein modification, there are often differences in the electrophoretic mobility of the same protein from different sources, resulting in unclear indications.

At the same time, these standard molecular weight proteins are often pre-stained in western hybridization. The difference in the binding firmness and binding amount between the dye and the protein molecular weight standard affects the mobility and indication effect of electrophoresis, and is expensive.

With the maturity of protein heterologous recombination expression technology, in order to make the purification of recombinant protein more convenient, more and more recombinant expression protein ends carry tags that are conducive to affinity purification, such as his-tag, etc. Electrophoretic detection of these proteins is often Relying on SDS-PAGE and western hybridization techniques, the protein molecular weight standards used in the analysis process often have many of the above-mentioned problems. At the same time, the protein molecular weight standards cannot be displayed simultaneously during the detection of the target protein, resulting in practical problems such as complicated operations. Therefore, the development of Protein molecular weight markers displayed synchronously with the target protein are of great value

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