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Application of pronucleus-derived ubiquitin-like molecules

A ubiquitin-like, prokaryotic technology, applied in the field of heterologous expression of recombinant proteins, can solve problems such as unknown sulfur transport molecules, and achieve the effects of increasing expression levels, increasing in vivo stability, and increasing the proportion

Inactive Publication Date: 2012-11-28
WEIZHI LANSI MEDICAL TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] However, it is not yet known whether the sulfur transport molecules MoaD and ThiS with ubiquitin-like structure in prokaryotic cells have ubiquitination modification function, and their feasibility as fusion partner expression systems for ubiquitin-like molecules of prokaryotic origin

Method used

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  • Application of pronucleus-derived ubiquitin-like molecules
  • Application of pronucleus-derived ubiquitin-like molecules
  • Application of pronucleus-derived ubiquitin-like molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. The effect of fusion partner ThiS or MoaD on the expression of mouse ribonuclease inhibitor mRI.

[0062] In order to observe the fusion partner effect of ThiS and MoaD and compare with ubiquitin and Sumo, the fusion expression vector Ub-mRI / pVI and Sumo-mRI / pVI of ubiquitin and Sumo and mouse ribonuclease inhibitor mRI were constructed. And MoaD and ThiS and mRI fusion expression vector MoaD-mRI / pVI and ThiS-mRI / pVI. The fusion expression products all contain an amino-terminal polyhistidine tag. mRI is a protein that is prone to degradation in vivo (Ge Ying, et al. Chinese Journal of Bioengineering 2010, 30: 17-23), which is convenient for observing the effects of fusion partners. In TG1 and BL21(DE3)pLysS strains induced by IPTG, Ub-mRI and Sumo-mRI mainly contain complete fusion expression products, and many low-molecular-weight degradation bands can be seen; in BL21(DE3) lacking Lon protease In pLysS bacteria, product degradation is reduced ( image 3 A-B)...

Embodiment 2

[0065] Example 2. Fusion expression of fusion partner ThiS and human insulin A chain.

[0066] Human insulin A chain is a small polypeptide containing 21 amino acid residues, which needs to be expressed heterologously in the form of a fusion protein. A chain protein expression vector ThiS-A / pET28a and Ub-A / pET28a fused with ThiS and ubiquitin were constructed to promote the expression of human insulin A chain and to compare the fusion partner effects of ThiS and ubiquitin. The theoretical calculated molecular weights of ThiS-A and Ub-A fusion proteins are 13438 and 14561, respectively. After BL21(DE3)pLysS IPTG induction, both of them produced protein bands of expected size without obvious degradation products. However, the yield of ThiS fusion products is significantly higher than that of ubiquitin fusion products ( Image 6 ).

Embodiment 3

[0067] Example 3. Fusion expression of fusion partner ThiS and human insulin B chain.

[0068] The human insulin B chain is a small molecule polypeptide containing 30 amino acid residues, which also needs to be expressed heterologously in the form of a fusion protein. The B-chain protein expression vectors ThiS-B / pET28a and Ub-B / pET28a fused with ThiS and ubiquitin were constructed to promote the expression of human insulin B chain and to compare the fusion partner effects of ThiS and ubiquitin. The theoretical calculated molecular weights of ThiS-B and Ub-B fusion proteins are 14484 and 15607, respectively. After BL21(DE3)pLysS IPTG induction, both of them produced protein bands of expected size without obvious degradation products. However, the yield of ThiS fusion products is significantly higher than that of ubiquitin fusion products ( Figure 7 ).

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Abstract

The invention relates to application of pronucleus-derived ubiquitin-like molecules, belonging to the technical field of bioengineering. The invention discloses an effectively method for enhancing expression level of a recombinant target protein. Pronucleus-derived ubiquitin-like molecules, especially sulfur transporters MoaD or ThiS, and the target protein are subjected to recombinant fusion expression to obtain the high-level target protein product.

Description

Technical field: [0001] The invention belongs to the field of bioengineering, and particularly relates to the heterologous expression of recombinant proteins. Background technique: [0002] Expressing heterologous genes in host cells, including bacteria, yeasts, mammalian cells and insect cells, is currently the most commonly used method for producing protein and / or polypeptide products. The use of heterologous expression systems to obtain uniform proteins with biological activity is also widely used in functional genomics research. Prokaryotic systems such as Escherichia coli have been widely used due to their simple bacterial cells and low cost of expressing proteins. [0003] However, for different heterologous genes, in the same host cell, although the same expression vector is used to obtain the same transcription and translation signal, the expression level of the recombinant protein can be very different. There are many reasons why certain recombinant proteins cannot be ex...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/63C07K19/00
Inventor 王楠许建葛莹
Owner WEIZHI LANSI MEDICAL TECH BEIJING
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