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E. coli expression system types

Update time : 2021-02-03

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E. coli expression system types
The E. coli expression system is the most widely used protein expression system. About 30% of the recombinant protein products on the market are produced by the E. coli system. Generalbiosystems' E. coli expression system roughly has three types of expression.

1. T7 E. coli expression

T7 E. coli expresses the target gene at a higher level. But if the target gene is toxic to E. coli, it will affect cell growth. T7 E. coli expression is an expression system constructed with T7 promoter and T7 phage transcription system elements. T7 RNA polymerase is a highly active RNA polymerase. 

The synthesis speed of mRNA is five times that of the RNA polymerase in E. coli. Some sequences that cannot be transcribed by E. coli RNA polymerase can also be transcribed. Therefore, T7 RNA polymerase and T7 In the presence of the phage promoter, the transcription system of E. coli itself cannot compete with the T7 phage transcription system, and ultimately the T7 phage promoter controls protein transcription.

2. Lac and Tac E. coli expression system

Lac and Tac Escherichia coli expression system is an expression system designed and constructed based on the regulation mechanism of Escherichia coli lac operon. The shellac operon has a polycistronic structure. In the absence of an inducer, the negative regulatory factor lacI gene product binds tightly to the operator downstream of the promoter and starts tissue transcription. 

In the presence of the inducer IPTG, after binding to the repressor, the ability to bind to the operon is reduced and dissociated, thereby activating the transcription of the lac operon. The expression system constructed with the tac promoter is called the Tac expression system. 

In order to strictly regulate the Lac and Tac expression systems, a mutant lacIq of the lacI gene that can produce excessive lacI repressor protein is applied to the expression system. However, when the expression vector was constructed using high-copy replicons, a higher level of background transcription was still observed, and the lacIq gene was inserted into the expression vector to ensure more lacI repressor protein production.

3. PL and PR E. coli expression system

The system constructed with the early transcription promoters PL and PR of lambda phage as the core is called PL and PR E. coli expression system. Promoters PL and PR have a strong ability to initiate transcription. 

The use of them to construct expression vectors has been proposed and studied in the early development of E. coli expression system. This expression system is based on the gene product of the temperature-sensitive mutant cl857 to regulate the transcription of the PL and PR promoters. 

The gene product exists in an active form at a lower temperature (30°C), and loses at a higher temperature (42°C). live. This expression vector is quite unstable in ordinary E. coli, which is caused by the high-strength direct transcription of the PL or PR promoter without the cl gene product in the bacteria. 

One way to solve this problem is to use lysogenic lambda phage Escherichia coli as the host bacteria for PL and PR promoter expression vectors; the second way is to assemble the cI857ts gene on the expression vector, so that it can be larger The range of host bacteria to choose from.

Since the PL and PR expression systems do not need to add chemical inducers during induction, and the cost is low, the first few medicinal recombinant proteins prepared in the E. coli system all use this expression system. However, it also has its own shortcomings. 

First of all, during the heat stimulation process, the expression of E. coli heat shock protein is also activated, including proteolytic enzymes, so it may degrade the expressed recombinant protein. 

Secondly, when large-volume fermentation and culture of bacteria, it is relatively slow to increase the culture temperature from 30°C to 42°C. This kind of heating method may affect the induction effect and the expression of recombinant protein.

For E. coli expression requirements, please contact Generalbiosystems——protein expression company.