Recombined ULP1 kinase coding sequence, coding protein, plasmid containing recombined ULP1 kinase coding sequence and recombined ULP1 kinase preparation method

A coding sequence and plasmid technology, applied in the field of molecular biology, can solve the problems of complex preparation process and low purity

Inactive Publication Date: 2016-01-27
NAT UNIV OF DEFENSE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In relevant foreign literature, although there are relevant reports, i

Method used

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  • Recombined ULP1 kinase coding sequence, coding protein, plasmid containing recombined ULP1 kinase coding sequence and recombined ULP1 kinase preparation method
  • Recombined ULP1 kinase coding sequence, coding protein, plasmid containing recombined ULP1 kinase coding sequence and recombined ULP1 kinase preparation method
  • Recombined ULP1 kinase coding sequence, coding protein, plasmid containing recombined ULP1 kinase coding sequence and recombined ULP1 kinase preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation process of recombinant ULP1 kinase

[0028] 1. According to the codon usage preference of E. coli, for ULP1 (403-621) The coding genes corresponding to this part of the protein were optimized. The specific optimized sequence is shown in SEQ ID NO.1, and a 6xHis tag is introduced at the end of the sequence.

[0029] 2. Carry out the whole gene synthesis of the sequence corresponding to SEQIDNO.1, and connect the synthesized SEQIDNO.1 into the commercial expression vector pCold-II (the vector is self-sustained at the front end of NdeI) by enzyme digestion (NdeI and HindIII) connection. with 6xHis sequence), named pCD-ULP1, and the sequence is shown in SEQ ID NO.2. like figure 1 As shown, at this time both N and C ends of ULP1 kinase have 6xHis tags (6xHis-ULP1 (403-621) -6xHis), the recombinant ULP1 kinase with this structure has a very high affinity to the nickel column, so it can be eluted with 200mM imidazole, and finally the recombinant ULP1 ...

Embodiment 2

[0038] Example 2 Effects of Different Temperatures on the Heterologous Expression of Recombinant ULP1 Kinase

[0039] The inventors also explored the expression of recombinant ULP1 kinase in Escherichia coli at different temperatures. It was found that the recombinant ULP1 kinase has a good expression effect at a very wide range of temperatures (16-37 degrees) ( figure 2 ), which means that researchers can choose the appropriate temperature according to the needs of the experimental progress. figure 2 shows the expression of recombinant ULP1 kinase at 16 degrees and 37 degrees, and found that at these two temperatures, recombinant ULP1 kinase can be clearly expressed. Simultaneously, the inventor also explored other temperatures such as 30 degrees, 25 degrees, 20 degrees, etc., and the results were basically consistent with 37 degrees. The lower the temperature, the slower the growth of the bacteria, so the induction time will be extended as the temperature decreases, and ...

Embodiment 3

[0040] Example 3 Activity Detection of Recombinant ULP1 Kinase

[0041] The inventors compared the cleavage efficiency of the recombinant ULP1 kinase under different conditions, such as image 3 As shown, it is found that the recombinant ULP1 kinase prepared by this method has very high activity in its cutting standard buffer, Tris-HCl, PBS buffer, and even pure water, and can completely cut the substrate protein. The standard reaction system is: total volume 50 μl, 5 μg substrate (sumo-eGFP), 1 μl prepared recombinant ULP1 kinase.

[0042] Standard cleavage buffer composition: 25mM Tris-HCl, 1% NP-40, 250mM NaCl, 50% (V / V) Glycerol, pH=8.0.

[0043] Tris-HCl buffer composition: 25mM Tris-HCl, pH=8.0.

[0044] PBS buffer composition: 38.7mMNa 2 HPO 4 ,11.3mMNaH 2 PO 4 , 150mM NaCl, pH=7.4.

[0045]

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Abstract

The invention discloses a recombined ULP1 kinase coding sequence, a coding protein, a plasmid containing the recombined ULP1 kinase coding sequence and a recombined ULP1 kinase preparation method. The recombined ULP1 kinase coding sequence, the coding protein, the plasmid containing the recombined ULP1 kinase coding sequence and the recombined ULP1 kinase preparation method have the advantages that a heterogeneous expression method combining an automatic induction medium with an expression vector containing cspA promoters is adopted, so that high-activity recombinant expression and high-purity enrichment of a ULP1 protein are achieved; when UPL1 gene expression is induced, OD600 monitoring is unneeded, and finally, the ULP1 protein with purity above 90% can be obtained by a one-step method; according to the method, stable expression effect on the ULP1 protein can be achieved within the temperature range of 16-37 DEG C, and the final yield is above 15 mg/L; the purified ULP1 protein is high in activity and capable of cutting a substrate effectively under general conditions.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a coding sequence of recombinant ULP1 kinase, a coded protein, a plasmid containing the coding sequence, and a preparation method of the recombinant ULP1 kinase. Background technique [0002] Fusion expression of polypeptides or proteins in E. coli is a very common technique, but in order to release the target polypeptide or protein from the fusion protein, it is often necessary to use expensive protein kinases or use intein self-cleavage. Intein self-cleavage has a preference for the first amino acid at the N-terminal or C-terminal of the target polypeptide, and the cleavage effect is unstable. Therefore, various protein kinases are currently used to cleave the fusion protein, thereby releasing the target polypeptide (protein ). [0003] Currently, common protein kinases that cleave fusion proteins include thrombin, factor 10 protein, enterokinase, TEV kina...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/70C12N9/12
Inventor 孟尔张东裔吴慧鲍邵衡李文颖吴磊朱凌云柳珑王干
Owner NAT UNIV OF DEFENSE TECH
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