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Clone and expression of bovine recombinant alpha-1,3-galactoside transferase (GT)

A technology of galactoside and transferase, applied in the field of recombinant enzyme protein, can solve problems such as low activity, inability to apply clinically, and complicated procedures

Inactive Publication Date: 2011-11-23
INNER MONGOLIA MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In 2001, Chen et al. (ChenAC, et al. Glycobiology 2001, 11:577-586) used a yeast expression system to produce GT with glycosylation. According to their reports, the activity was high, but when we repeated their experiments It is found that it takes a long time and the cost is high, especially when there are always many kinds of recombinant protein contamination during purification. Although after repeated purification, there are still a large number of glycosylated enzyme proteins with different degrees of contamination
[0007] In a nutshell, the existing GT production methods are divided into the following categories: 1) extract from bovine thymus, but the procedure is complicated, the yield is low, and the activity is also low; Genes are expressed in prokaryotic cells, but we found that the activity of the enzyme protein they express is not high, so they cannot be used clinically; 3) the application of eukaryotic expression system, although the expression is glycosylated protein, but the expression level is low, the expression required Long time and high cost
In addition, some people use insect cell systems for expression, but the yield is low and cannot be applied clinically

Method used

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  • Clone and expression of bovine recombinant alpha-1,3-galactoside transferase (GT)
  • Clone and expression of bovine recombinant alpha-1,3-galactoside transferase (GT)
  • Clone and expression of bovine recombinant alpha-1,3-galactoside transferase (GT)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Construction of expression recombinant bovine α-1,3-GT plasmid

[0024] The invention adopts the amino acid from the 80th to the 368th amino acid of the Inner Mongolia cow thymus GT protein, which is the smallest water-soluble part with GT activity. In order to further improve the water solubility and enzyme reactivity of recombinant GT, through the structural analysis of the enzyme protein, the two amino acids at the 160th and 241st positions have a greater impact on the activity and water solubility of the enzyme, so the present invention uses GT at the 160th Amend amino acid proline (P) to lysine (K); modify glutamic acid (E) at position 241 to glutamine (Q), see figure 1 .

[0025] First extract mRNA from bovine vascular endothelial cells, and amplify the bovine α-1,3-GT cDNA fragment through reverse transcription PCR (RT-PCR). The position is at 1104bp (namely at the 368th amino acid). The cDNA fragment amplified by reverse transcription PCR was clone...

Embodiment 2

[0026] Example 2. Purification of recombinant GTase protein

[0027] HMS174(DM3)E.coli containing pET-15b+α-1,3-GT plasmid was cultured to OD in LB medium 600 When = 0.7, add a final concentration of 1mM IPTG to induce the expression of the recombinant protein, continue to culture the bacteria at 35°C for 4 hours, centrifuge and collect the bacteria; freeze them on dry ice for 1.5 hours, then suspend them in the His-tag purification solution, and collect the supernatant by centrifugation , purified twice according to the His-tag purification column of Sigma Company, dialyzed three times in a cold storage at 4°C, and finally removed endotoxin with a kit. The purity of the expressed protein was determined by SDS-PAGE electrophoresis and high performance liquid phase, and quantified.

Embodiment 3

[0028] Embodiment 3. Activity test of recombinase protein

[0029] 3.1 Isotope test for α-1,3-GT enzyme activity

[0030] Test principle (see image 3): 100 μL containing 5 mM MnCl 2 , 5mM Tris-HCl (pH7.0), 50μM UDP-Gal (labeled isotope) and 10mM sugar acceptor Galβ-1, 4-GlcNAcβ-OR, added different concentrations of recombinant GT, reacted at 37°C for 15 minutes, added 20 μL ice water to stop the reaction. Add 0.05ml of the reaction solution into a liquid scintillation vial of AG1-X8 anion exchange resin (Bio-Rad), and measure the isotope after repeated washing.

[0031] Quantitative standard of enzyme activity: 1 unit of enzyme = transfer of 1 μmol Gal from UDP-Gal to Galβ-1,4-GlcNAcβ-OR sugar acceptor within 15 minutes at 37°C.

[0032] Test Results:

[0033] A. The expression amount of enzyme protein of the present invention: >10 mg of enzyme protein can be produced per liter of LB culture solution; enzyme activity: 15 units / mg. Electrophoresis confirmed that the mol...

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Abstract

The invention relates to a recombinant zymoprotein having high activity and specific GT. According to the invention, the 80th to 368th amino acids of Inner Mongolia cow thymus GT proteins, the smallest water-soluble part with activity of GT, are employed. To further improve water-solubility and enzyme reaction activity of recombinant GT, through molecular designing and gene engineering reconstruction of natural proteins, the 160th amino acid of GT proline is modified to be lysine, the 241st amino acid of GT glutamic acid is modified to be glutamine, and therefore, recombinant zymoprotein withstronger enzymatic activity is obtained; the obtained recombinant zymoprotein has enzymatic activity 8 to 10 times that of existing zymoprotein and is applicable to the medical field of tumor treatment.

Description

technical field [0001] The invention relates to a highly active recombinant enzyme protein with specific galactosidyl transferase (GT) activity. The enzyme protein is a recombinant enzyme protein with stronger enzyme activity obtained through molecular design and genetic engineering of natural proteins , the recombinant enzyme protein can be applied to the medical field of treating tumors. Background technique [0002] Organ transplantation is an important treatment technology in contemporary medicine, but the widespread development of this medical technology has been seriously affected due to the shortage of human organs. In order to find individual organs, pigs may be a good candidate for interspecific transplantation. However, if humans accept pig-derived donors, antibody-mediated hyperacute rejection is a major obstacle. The main reason for the rejection of this heterogeneous transplantation is due to the galactosyl chain on the surface of porcine vascular endothelial ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/00A61P35/00
Inventor 云升邱英
Owner INNER MONGOLIA MEDICAL COLLEGE
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