Recombinant Protein Expression Introduction

1. What is Recombinant Protein Expression？

It reveals that life activities can be explored from different levels such as genes and proteins. With the continuous development of science and technology, protein research has developed from protein extraction to recombinant protein expression. At present, we can use a variety of recombinant protein expression systems (prokaryotic expression system, yeast expression system, insect expression system, mammalian expression system, plant expression system) to recombinant expression, separation and purification of our target proteins to further explore the function of proteins, providing a convenient method for scientific research.

2. Experimental Procedure of Recombinant Protein Expression

The main components include expression vectors and host cells, and the main experimental procedures are as follows:

(1) Construction of expression vector

The selection of expression vector is usually based on the purpose of the expressed protein, known information and cloning and purification strategies. After the vector is selected, the target gene is connected to the expression vector by homologous recombination or seamless cloning.

(2) Transform host cells

Host cell selection can be based on the characteristics of the host strain as well as the use of the target gene expression product. For example, when there are more proteases produced in the host cell, which affects the stability of the target gene expression product, the protease-deficient strain can be selected, such as BL21 series is suitable for the expression of toxic proteins. When the foreign gene to be expressed is a eukaryotic gene, which is difficult to be expressed in prokaryotes, the Rosetta series strains supplemented with rare codons can be selected. After selecting a suitable host strain, the constructed plasmid containing the target gene is transformed into the host cell, and the positive clone is screened.

(3) Induced expression of target protein

Culture conditions are the key factors for successful production of recombinant proteins. IPTG is the commonly used induction method for prokaryotic expression, and methanol is the commonly used induction method for yeast expression. However, exploration and optimization should be carried out for different target genes and host strains, the concentration of inducers, and the culture temperature and time to achieve the best culture conditions.

(4) Separation and purification of target protein

According to the properties of proteins, different expression forms and fusion labels, suitable methods were selected to isolate and purify the target proteins.

(5) Detection and identification of target protein

The size and purity of purified protein can be detected by SDS-PAGE and Western-blolt methods. All procedures should be performed on ice to prevent protein denaturation.

3. Several Types of Recombination Expression

According to the location of protein expression and secretion, protein expression can be divided into intracellular expression and secretory expression, mainly intracellular expression. According to the product form of protein, it can be divided into soluble expression and insoluble expression (inclusion body expression). According to the expression method of target protein, the expression forms of target protein can be divided into fusion expression and non-fusion expression.

(1) Fusion expression

Fusion expression refers to the method that the gene sequence of the target protein and other protein tag gene sequences are constructed into the expression vector and controlled by the same regulatory elements for fusion expression. This method has the following characteristics: fusion expression can improve the expression efficiency of the target protein, help the protein to fold correctly, facilitate soluble expression, and the expressed protein is relatively stable. In addition, protein tags such as His are used to make purification easier, and the purified recombinant protein can be removed from the protein tag in vitro.

(2) Non-fusion expression

Non-fusion expression, as opposed to fusion expression, means that the target protein is not fused with any protein label, but the protein expressed in this expression mode is often degraded by the protease in the host, and the expressed protein cannot be correctly folded and lose biological activity.

(3) Intracellular expression

The expression forms of most target proteins are mainly intracellular, and sometimes intracellular expression often has a certain impact on the structure of proteins. For example, during prokaryotic expression, the intracellular expression of proteins is often prone to excessive intracellular expression, and the proteins are not properly folded and modified in time, thus forming inclusion bodies and losing protein activity. Moreover, in yeast, the expression of intracellular proteins is often low, and it is easy to be over-glycosylated by yeast. Therefore, during protein expression, we can use some protein tags such as target proteins or secreted signal peptides to help the recombinant proteins to secrete and express.

(4) Secretory expression

The expression form of recombinant protein secreted outside the cell is called secretory expression, which is very easy to purify the protein, and the protein is relatively stable and not easy to degrade.

(5) Soluble expression

Since only a few proteins can be secreted and most proteins are mainly expressed intracellular, the methods of fusion expression, inclusion body renaturation and reduction of protein expression speed can be used to make proteins express in soluble form.

(6) Insoluble expression (inclusion body expression)

Insoluble expression means that when the protein is expressed at a high level in the host body, the solid particle precipitation is formed due to the failure to properly fold in time, which is called inclusion body expression, so insoluble expression is also called inclusion body expression. Inclusion bodies are often not biologically active because they do not have the correct structure, but their amino acid sequence is correct. The reason of inclusion body formation is mainly related to the nature of protein itself, culture conditions and molecular chaperones.

Therefore, when solving the inclusion body problem, the common solutions include adding protein tags for fusion expression/secretion expression, adjusting the induction conditions (such as reducing the culture temperature, selecting the appropriate promoter, inducer, etc.), reducing the protein expression rate, and reducing the inclusion body renaturation.

KMD Bioscience has been committed to the expression, separation and purification of recombinant proteins for many years, and has built protein platforms such as prokaryotic expression system, yeast expression system, insect expression system, mammalian expression system and plant expression system for different protein expression needs. For protein expression purification, we can provide the following services and protein expression solutions.

(1) Different expression systems are provided according to the properties of proteins

(2) Protein soluble expression

Based on the analysis of protein properties, we can recommend an optimal expression scheme and expression system. We have a variety of protein labels, such as GST, His, SUMO, etc., which can help protein soluble expression, and we have independently developed and designed and modified a variety of carriers, which can carry out protein expression in a short time. In addition, our company has a mature protein purification platform, which can purify proteins through a variety of protein purification methods, and can also remove protein labels for recombinant proteins in vitro. At the same time, we have a unique inclusion body revival technique that allows us to experiment with renaturation of inclusion bodies.

(3) It has a variety of protein expression systems to meet the different needs of scientific research and industrial users.

(4) Strict quality control standards: SDS-PAGE and WB quality detection of purified protein.

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