1. What is Prokaryotic Expression?

The method of using gene cloning technology to convert foreign target genes into host cells with the help of expression vectors and make them stably expressed in prokaryotic organisms is called prokaryotic expression.

2. The Principle of Prokaryotic Expression

2.1 Basic Composition of Prokaryotic Expression

The prokaryotic expression system consists of two parts: the expression vector and the host cell.

An expression vector is a recombinant DNA molecule consisting of promoters, screening tags, polyclonal sites, replicons, replication initiations and fusion tags. Promoters are divided into strong and weak, not all genes are suitable for strong promoters and some genes are rapidly expressed in large quantities driven by strong promoters, which often causes their translation products to not fold correctly and form inactive inclusion bodies. Fusion tags can help protein expression and purification, common fusion tags include His, GST, HA, FLAG, etc.

Host cells refer to organisms that can achieve the transcription, translation and synthesis of target proteins, and common prokaryotic expression hosts include Escherichia coli and Bacillus subtilis.

2.2 Experimental Procedure for Prokaryotic Expression

(1) Expression Vector Construction

The use of expression vectors is often selected based on the purpose of the protein being expressed, known information, and cloning and purification strategies (KMD Bioscience can provide expression vectors with various fusion tags to promote protein solubility and simplify protein purification.). After selecting the vector, the target gene can be constructed onto the expression vector by homologous recombination or seamless cloning.

(2) Transform Host Cells

The choice of host bacteria is determined by the host bacteria and foreign gene expression products, and the host bacteria commonly used for prokaryotic expression are Escherichia coli (gram-negative bacteria) and Bacillus subtilis (gram-positive bacteria). It is usually considered according to the following factors: ① Whether the host bacterial endogenous enzyme will affect the stability of the expressed protein, E. coli usually produces endotoxin, while Bacillus subtilis does not produce endotoxin; the common BL21 series is lon and ompT protease deficient strains. ② Host bacterial codon preference, eukaryotic cell and prokaryotic cell codon preference are different, when expressing eukaryotic genes can choose the Rosetta series. ③ Whether the expressed protein needs to be folded, K-12 derived bacteria Origami 2 series can promote the formation of disulfide bonds, help the protein to fold correctly and improve the solubility and activity of the protein. After selecting the appropriate host bacteria, the recombinant plasmid containing the gene of interest is transformed into the host bacterial cells and screened for positive clones.

KMD has collected and established a variety of strains including a cryogenic inductive expression to choose from, please visit the company's website for further information.

(3) Induced Expression of the Protein of Interest

IPTG induction is a common induction method. Low concentrations of IPTG (0.2-0.5mM) do not reduce the expression amount but can increase the solubility of the protein. Too high an IPTG concentration can speed up protein expression, resulting in translation products that cannot be folded correctly and contain bodies. During the experiment, it is necessary to continuously optimize the optimal concentration of IPTG to achieve the best activity and solubility of the target protein. In addition, the temperature of the culture also has a certain influence on the soluble expression of the protein and the optimal temperature and incubation time should be constantly explored in the experiment.

(4) Isolation and Purification of the Protein of Interest

The separation and purification of the target protein is divided into three steps: pretreatment, coarse separation and fine separation.

Pretreatment: The process of performing preliminary processing of a sample, releasing proteins from the sample and maintaining their original state.

Crude separation: The purpose of this step is to purify the protein of interest by salting out, isoelectric precipitation and organic solvent fractionation to separate it from other heterogeneous proteins.

Fine separation: Generally, the corresponding purification method is selected according to the tag for further purification of the protein of interest.

(5) Detection of the Protein of Interest

The protein of interest can be detected by SDS-PAGE, Western blot and ELISA. It is important to note that to prevent protein denaturation, all manipulations should be performed on ice.

Figure 1 Prokaryotic Expression Procedure Diagram

3. Fusion Tags and Expression Host Types

Table 1 Main Characteristics of Protein Tags

Table 2 Host System Selection

4. Advantages and Disadvantages of Prokaryotic Expression Technology

Advantages:

(1) The genetic background is clear.

(2) High efficiency: high expression level of the target gene and short culture cycle.

(3) Simple operation, easy to grow and control.

(4) Low cost, suitable for large-scale production.

(5) There are abundant strains and supporting carriers to choose from.

Disadvantages:

(1) The expressed protein is not necessarily active without modification.

(2) Translation products are often expressed in the form of inclusion bodies.

(3) Endotoxin is produced.

5. Application

Prokaryotic expression is widely used in the R&D and production of antibodies, vaccines, drugs, animal and plant growth regulators, and anti-insect proteins. In addition, prokaryotic expression is also used in the production process of industrial enzymes and biological dyes with its high efficiency and low cost.

6. What Kind of Prokaryotic Expression Services Can KMD Provide?

KMD Bioscience has a complete prokaryotic expression purification system, which can provide customers with a variety of expression vectors and host strains, can provide customers with a high-quality expression strategy, and we have a wealth of purification methods to ensure a high-quality purification program. Customers only need to provide us with a protein sequence, CDS or protein name, we can complete the expression and purification of the target protein for you in a short time, and provide you with a one-stop service.