Lyase of self-cleaving escherichia coli, and applications thereof

A technology of Escherichia coli and lyase, applied in the field of microorganisms, can solve the problems of refractory host bacteria, inability to realize controllable self-cleavage of Escherichia coli, and conservation of lyase action sites, etc.

Active Publication Date: 2015-07-08
菲吉乐科股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lyase has high specificity and can only specifically recognize and lyse specific types of bacteria
In addition, the action site of lyase is very conserved, coupled with the specificity of co-evolution between phage and bacteria, it is difficult for host bacteria to develop resistance to it
So far, some natural lysing enzymes that can lyse E. coli in vitro have been reported. These enzymes can lyse E. coli directly or with the assistance of membrane permeabilizing agents (such as EDTA), but cannot achieve E. coli when expressed in vivo. Controlled self-lysis of bacilli
At present, there is no report on the use of lyase to realize the controlled self-lysis of E. coli for exogenous protein production

Method used

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  • Lyase of self-cleaving escherichia coli, and applications thereof
  • Lyase of self-cleaving escherichia coli, and applications thereof
  • Lyase of self-cleaving escherichia coli, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, the construction of the lyase that can induce Escherichia coli self-cleavage

[0022] (1) Synthesized in Nanjing GenScript Biotechnology Co., Ltd., which can express the cleavage enzyme ClyN ClyN genetic DNA sequence. The synthetic sequence was loaded into the pUC57 plasmid. by ClyN The gene is used as a template, and NcoI and XhoI restriction sites are added to the two ends of the target gene respectively. The primers are designed as follows:

[0023] Forward primer: 5-ATAT CCATGG GCATGCCTCCATCATTACG-3 (SEQ.ID.NO.3)

[0024] NCOI

[0025] Reverse primer: 5-TATA CTCGAG GTAGTTCAGGGTCTGGCCTG-3 (SEQ.ID.NO.4)

[0026] wxya

[0027] Using 2 μl of the gene as a template, add 1 μg of primers for PCR amplification. The PCR amplification procedure is as follows: 1) 94 °C, 5 min; 2) 94 °C, 30 sec, 62 °C, 45 sec, 72 °C, 45 sec , 30 cycles; 3) 72 ℃, 10 min; the product was recovered by gel electrophoresis, and the electrophoresis pattern was as follows: f...

Embodiment 2

[0029] Example 2, Verification of ClyN-induced Escherichia coli self-lysis

[0030] Culture Escherichia coli BL21(DE3) / pET-ClyN to OD 600 =0.8, after adding IPTG to 0.5 mM, use a microplate reader to monitor the change of the absorbance value of the bacterial solution at 600 nm. At the same time, the bacterial solution without IPTG was used as a negative control. The final pyrolysis curve obtained is shown in the appendix figure 2 . The results showed that ClyN could induce rapid self-lysis of the host strain resulting in a rapid decrease in absorbance at 600 nm.

Embodiment 3

[0031] Example 3, Verification of the results of changes in ATP in the culture medium after ClyN-induced Escherichia coli self-lysis

[0032] Culture Escherichia coli BL21(DE3) / pET-ClyN to OD 600 =0.8, after adding IPTG to 0.5 mM, use the luciferase detection kit to detect the change of the luminescence value in the bacterial solution. At the same time, the bacterial solution without IPTG was used as a negative control. The luminescence curve obtained from the test is shown in the appendix image 3 . The results showed that a large amount of ATP could be detected in the culture medium after ClyN induced the self-lysis of the host Escherichia coli.

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PUM

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Abstract

The invention discloses a lyase of self-cleaving escherichia coli, and applications thereof. A brandnew lyase ClyN is constructed by adopting gene splicing means, the lyase can result in the self-cleaving of the host escherichia coli after being expressed in the escherichia coli, to release intracellularly expressed ATP and protein. Recombinant ClyN expression plasmid can be duplicated in the escherichia coli BL21(DE3), and express for 15 min under the induction of IPTG to observe the obvious phenomenon of inducing the escherichia coli to self-cleave, and the induced cleaving efficiency achieves 99.8%. When the host bacteria express heterologous enhanced green fluorescent protein (EGFP), EGFP can be released in solution after escherichia coli is cleaved, and the release efficiency is 89% of direct ultrasonic breaking efficiency. Therefore, the ClyN can be used for extracting and release of heterologous protein after expression in the escherichia coli, thus having wide application prospects.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a lyase for internally lysing Escherichia coli and an application thereof. Background technique [0002] Escherichia coli is a kind of Gram-negative bacteria, because its genetic background is relatively clear, it is a commonly used model strain in molecular biology, and it is also a very important engineering strain in industry. Escherichia coli is a production strain of many foreign proteins, and is often used to induce the production of various proteins, such as fluorescent protein (EGFP), succinic acid, Harpin protein, interferon, etc. In order to obtain the exogenous recombinant protein expressed in Escherichia coli, it is usually necessary to collect the host cells by centrifugation, and then disrupt them by ultrasonication or pressure. This requires large instruments and equipment, but also consumes a lot of energy. Therefore, it is of great significance to find...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70
CPCC12N9/88
Inventor 危宏平杨航余军平
Owner 菲吉乐科股份有限公司
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