Precursor protein capable of producing multiple effective constituents after cutting

A technology of precursor protein and amino acid, applied in the direction of animal/human protein, interleukin, depsipeptide, etc., can solve the problems of increased time and other costs, long fermentation time, long time of high temperature retention of interleukin 11, etc.

Inactive Publication Date: 2005-09-14
许日山
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another method is to use methanolic yeast to directly express recombinant human interleukin-11 (see patent 99118279.0). In this method, interleukin-11 is directly expressed in methanolic yeast medium, which facilitates downstream purification, but due to its fermentation time Longer (greater than 3 days), increased time and other costs, and the longer time for the expressed interleukin 11 to stay in the fermentation broth at high temperature also increased some unfavorable factors

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0024] The target gene was amplified from the human hepatocyte library by PCR method, and the first 10 amino acids (His-Pro-Pro-Lys-Ser-Aso-Leu-Val-Pro-Arg) were introduced and inserted into the restriction site when synthesizing primers point, and then use the sequencing plasmid pBSK to screen out the complete target gene fragment for later use, and finally cut out the sequence from the recombinant pBSK plasmid to determine the correct target gene segment, and directionally insert it into the expression plasmid pET21b to obtain the recombinant plasmid pET-pro, after enzyme digestion , sequenced, and identified recombinant plasmid pET-pro was transferred into the expression strain BL21(DE3) by the CaCl2 method, and the recombinant engineered strain was expressed and purified to obtain the final interleukin-11 protein mutant.

[0025] The amino acid sequence of the precursor protein is:

[0026] His-Pro-Pro-Lys-Ser-Aso-Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Val-Ser-Pro-Asp-Pro-Arg-

...

example 2

[0055] Using the PCR site-directed mutagenesis method, the 15th amino acid of the precursor protein in Example 1 was mutated to Ala, and the target gene fragment was excised from the recombinant pBSK plasmid to determine the correct sequence, and embedded in the expression plasmid pBV in a directional manner to obtain the recombinant Plasmid pBV-pro, the recombinant plasmid pBV-pro after digestion, sequencing and identification, was transformed into expression bacteria DH5α by CaCl2 method, and the recombinant engineering bacteria were expressed and purified to obtain the final interleukin-11 protein mutant.

[0056] The amino acid sequence of the precursor protein is:

[0057] His-Pro-Pro-Lys-Ser-Aso-Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser-Pro-Asp-Pro-Arg-

[0058] Ala-Glu-Leu-Asp-Ser-Thr-Val-Leu-Leu-Thr-Arg-Ser-Leu-Leu-Ala-Asp-Thr-Arg-Gln-Leu-

[0059] Ala-Ala-Gln-Leu-Arg-Asp-Lys-Phe-Pro-Ala-Asp-Gly-Asp-His-Asn-Leu-Asp-Ser-Leu-Pro-

[0060] Thr-Leu-Ala-Met-Ser-Ala-Gly-Ala-...

example 3

[0086] Using the PCR site-directed mutagenesis method, the 139th amino acid of the precursor protein in Example 2 was changed to Asn, and the target gene fragment was cut out from the recombinant pBSK plasmid to determine the correct sequence, and embedded in the expression plasmid pKK in a directional manner to obtain a recombinant plasmid pKK-pro, the recombinant plasmid pKK-pro after digestion, sequencing and identification, with CaCl 2 Transformed into the expression strain BL21 by the method, the recombinant engineered strain was expressed and purified to obtain the final interleukin-11 protein mutant.

[0087] The amino acid sequence of the precursor protein is:

[0088] His-Pro-Pro-Lys-Ser-Aso-Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser-Pro-Asp-Pro-Arg-

[0089] Ala-Glu-Leu-Asp-Ser-Thr-Val-Leu-Leu-Thr-Arg-Ser-Leu-Leu-Ala-Asp-Thr-Arg-Gln-Leu-

[0090] Ala-Ala-Gln-Leu-Arg-Asp-Lys-Phe-Pro-Ala-Asp-Gly-Asp-His-Asn-Leu-Asp-Ser-Leu-Pro-

[0091] Thr-Leu-Ala-Met-Ser-Ala-Gly-Ala-...

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Abstract

This invention relates to precursor albumen that can generate multiple kinds effective contents through cutting. Stable and high efficiency expression of the precursor albumen in colibacillus genetic engineering can be got by this invention. Extra high efficiency bioactivity effective constituent albumen is got after the expressed precursor albumen is cut. It can be used as accessory cure of cancer.

Description

1. Technical field [0001] The invention belongs to the field of recombinant protein medicine. Through the genetic engineering of the precursor protein, high-efficiency expression has been achieved; through the amino acid changes at different sites of the precursor protein and the control of the thrombin digestion process, a variety of mutants of mature interleukin 11 can be obtained. This method has the advantages of The expression level is high, the operation is simple, and the activity of the mature protein is high. 2. Background technology [0002] The precursor protein in the present invention is interleukin 11 precursor protein. Interleukin-11 is a type of cytokine with multiple functions in the human body. Its main functions are: (1) to differentiate hematopoietic stem cells into megakaryocytes, to proliferate and mature megakaryocytes, and to promote platelet production; (2) to protect Damaged cells of gastrointestinal mucosa; (3) promote the formation of bone marro...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K14/54C12N15/09
Inventor 许日山
Owner 许日山
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