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Construction method and application of Escherichia coli cold shock assistant dissolving type expression plasmids

A technology of Escherichia coli and expression plasmids, which is applied in the field of bioengineering to achieve good solubility, improved solubility, and good stability

Active Publication Date: 2018-09-07
HUNAN DANWEI BILOGICALTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Although researchers have applied a variety of techniques to recombinant protein expression, there is no universally applicable method for preparing soluble, homogeneous proteins with native N-termini

Method used

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  • Construction method and application of Escherichia coli cold shock assistant dissolving type expression plasmids
  • Construction method and application of Escherichia coli cold shock assistant dissolving type expression plasmids
  • Construction method and application of Escherichia coli cold shock assistant dissolving type expression plasmids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Construction of Escherichia coli Cold Shock Solubilizing Expression Vector

[0044] 1. Construction of ampicillin-resistant pCold-SUMOa plasmid

[0045] With the pE-SUMOpro Kan plasmid of LifeSensors company as a template, primers SUMO-F (sequence shown in SEQ ID No.3) and SUMO-R (sequence shown in SEQ ID No.4) were used to amplify the Smt3 fusion protein sequence ( The sequence is shown in SEQ ID NO.19). At the same time, using the pCold TF plasmid digested by Sal I as a template, the primers pCold-F (sequence shown in SEQ ID No.1) and pCold-R (sequence shown in SEQ ID No.2) were amplified to obtain pCold plasmid backbone fragment. Then adopt the seamless cloning technique to connect the above two fragments to obtain a recombinant plasmid, which is called pCold-SUMOa (such as figure 1 shown). Since the Smt3 fusion protein gene sequence contains two restriction sites, EcoR I and Pst I, the pCold TF plasmid after single digestion with SalI was selected as ...

Embodiment 2

[0050] Example 2. Construction of recombinant plasmid expressing cysteine ​​desulfurase

[0051] 1. Construction of a recombinant plasmid expressing Escherichia coli cysteine ​​desulfurase

[0052] Using the Escherichia coli E.coli MC4100 genome as a template, the primers IscS-pCold-F (sequence shown in SEQ ID No.9) and IscS-pCold-R (sequence shown in SEQ ID No.10) were amplified to obtain Escherichia coli IscS coding box gene. The pCold I plasmid was digested with Kpn I, and the digested vector fragment was recovered by gel. Then, the seamless cloning kit was used to construct the recombinant plasmid IscS-pCold I, and the recombinant plasmid was sent to BGI for sequencing identification.

[0053] 2. Construction of recombinant plasmid expressing human cysteine ​​desulfurase

[0054] With the NFS1(55-457)-pET28b plasmid as a template, primers m-NFS1-pCold-F (sequence shown in SEQ ID NO.11) / SUMO-m-NFS1-F (sequence shown in SEQ ID NO.13) were used ) and m-NFS1-pCold-R (seque...

Embodiment 3

[0111] Example 3. Induction and expression condition optimization of cysteine ​​desulfurase

[0112] Pass the recombinant plasmid constructed above through cold CaCl 2 Transformed into BL21(DE3) competent cells. The induction and expression of the recombinant cysteine ​​desulfurase of the present invention, unless otherwise stated, are all operated according to the following experimental steps:

[0113] (1) Inoculate the screened transformant overnight bacteria into LB liquid medium containing 100 μg / ml Amp at a volume ratio of 1:50, and culture with shaking at 37°C and 250rpm;

[0114] (2) When the OD600 value of the bacterial solution is about 0.4-0.5, immediately place the bacterial solution in an ice-water bath for 5 minutes, then move it to 15°C and let it stand for 30 minutes;

[0115] (3) Add IPTG to a final concentration of 100 μM / L, shake and culture at 15° C. and 250 rpm for 24 hours.

[0116] (4) After the culture is completed, use SDS-PAGE to analyze the presenc...

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Abstract

The invention relates to a construction method of Escherichia coli cold shock assistant dissolving type expression plasmids, and a method for preparing a water-soluble heterogenous polypeptide by applying the Escherichia coli cold shock assistant dissolving type expression plasmid. The cold shock assistant dissolving type expression plasmids for realizing small ubiquitin modified protein (SUMO) and exogenous gene fusion expression under the control of a promoter of a cold shock gene is constructed by using a seamless cloning technology. A chimeric cysteine desulfurase is cloned into the plasmids, such as widely-known pCold I and PET28 series plasmids, to obviously improve the water solubility, the stability and the enzyme activity of recombinant proteins. The SUMO has no influences on thetarget protein space conformation of widely-known pCold TF plasmids. The cutting efficiency of an SUMO label is more than 95% when enzyme digestion is performed at 25 DEG C for 1 h according to a ratio of Ulp1 protease to the recombinant protein of 1 U : (0.5-1) mg, so the Escherichia coli cold shock assistant dissolving type expression plasmids are very suitable for preparing proteins having natural N ends in the fields of bioengineering pharmacy and structural biology.

Description

technical field [0001] The invention relates to a method for constructing an Escherichia coli cold-shock soluble expression plasmid and a specific embodiment of its application, belonging to the technical field of bioengineering. Background technique [0002] Escherichia coli expression system, as the most commonly used recombinant protein expression system, has the characteristics of fast, mature technology, affordable, high yield and clear genetic background of strains. According to statistics, at present worldwide, more than 60% of the published literatures involving the expression of recombinant proteins are expressed in Escherichia coli, and nearly 30% of the medicinal proteins are produced by the expression system of Escherichia coli. However, when E. coli is used to express eukaryotic proteins, only about 30% of the cloned genes can be expressed in soluble form in E. coli, and others are easily degraded by E. coli protease, form inclusion bodies or cannot be expressed...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N9/50C12N15/54C12N9/10
CPCC12N9/13C12N9/50C12N15/70C12Y208/01007
Inventor 庞一林谭国强吕建新李江辉杜璟张涛孙倩倩韩琴霞
Owner HUNAN DANWEI BILOGICALTECH
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