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Novel human keratinocyte growth factor mutant

A keratinocyte and growth factor technology, applied in growth factors/inducing factors, animal/human proteins, peptides/protein components, etc., can solve problems such as reducing the biological activity and stability of expressed proteins

Inactive Publication Date: 2007-04-18
CHONGQING FAGEN BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of cysteine ​​containing no intramolecular disulfide bond is easy to form intermolecular disulfide bond in the expression of recombinant protein, that is, homodimer, which will significantly reduce the biological activity and stability of the expressed protein

Method used

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  • Novel human keratinocyte growth factor mutant
  • Novel human keratinocyte growth factor mutant
  • Novel human keratinocyte growth factor mutant

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0016] Example 1: Human keratinocyte growth factor (KGF) whole gene artificial synthesis scheme

[0017] According to the human KGF amino acid sequence (composed of 163 amino acids) reported in GenBank and literature, the full gene sequence of Δ23KGF was artificially synthesized by recursive PCR method, and the Nde I and EcoR I sites contained in it were changed. Escherichia coli or yeast preferred codons were selected for artificial synthesis in order to increase the level of recombinant expression.

[0018] Seq1: amino acid sequence of human KGF

[0019] cndmt peqma tnvnc ssper htrsy dymeg gdirv rrlfc rtqwy lridk rgkvk gtqem

[0020] 30 60

[0021] knnyn imeir tvavg ivaik gvese fylam nkegk lyakk ecned cnfke lilen hynty

[0022] 90 120

[0023] asakw thngg emfva lnqkg ipvrg kktkk eqkta hflpm ait

[0024] 150

[0025] Seq2: cDNA sequence of artificially synthesized human Δ23...

example 2

[0051] Example 2: Construction of the recombinant plasmid of the Δ24KGF (102S) mutant

[0052] Δ24KGF (102S) is a novel KGF mutant in which the cysteine ​​at position 102 is mutated to serine on the basis of the deletion of 24 amino acids at the N-terminal of human KGF. Taking the artificially synthesized human KGFcDNA fragment in Example 1 as a template, two pairs of primers were synthesized for PCR-guided directed point mutation and deletion.

[0053] Seq3: (forward) 5'-CATATGTACGACTACATGGAAGGTGGTGAC-3' (for N-terminal deletion)

[0054] Seq4: (reverse) 5'-TTCGTTGGATTCTTTTTTAGCGTA-3' (for 102-site mutation)

[0055] Seq5: (forward) 5'-GCTAAAAAAGAATCCAACGAAGAC-3' (for 102-site mutation)

[0056] Seq6: (reverse) 5'-GGATCCTCAGGTGATACCCATCGGCAG-3'

[0057] A 240bp fragment was amplified by PCR with Seq3 and Seq4 primers, and a 200bp fragment was amplified by PCR with Seq5 and Seq6 primers. Then, the 240bp and 200bp fragments were mixed, and PCR amplification was carried out ...

example 3

[0059] Example 3: Construction and expression of the recombinant plasmid of the Δ23KGF (25(2) mutant

[0060] Δ23KGF (25C) is a variant in which the 25th tyrosine is mutated into cysteine ​​based on the deletion of 23 amino acids at the N-terminal of human KGF. According to the method for example 2, synthesize following two pairs of primers:

[0061] Seq7; (Forward) 5'-CATATGTCTTGCGACTACATGGAAGGTGGTGAC

[0062] Seq8: (reverse) 5'-GGATCCTCAGGTGATAGCCATCGGCAG-3'

[0063] Using the artificially synthesized human KGF cDNA of Seq2 as a template, the fragment amplified by PCR was digested with Nde I and BamH I, and then inserted into the pET3a expression plasmid. After recombinant screening, transformation and expression screening, a strain expressing recombinant Δ23KGF (25C) was obtained.

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Abstract

A novel human keratinocyte growth factor mutant features that the cysteine at its site No.102 is mutated to become non-polar amino acid and the deficient mutation takes place at its terminal N. It can prevent the generation of scar and fibrosis and promote the epiderm healing.

Description

technical field [0001] The invention relates to a genetically engineered recombinant protein, and relates to a novel human keratinocyte growth factor mutant. The present invention also relates to the gene encoding the protein. Background technique [0002] Keratinocyte Growth Factor (KGF) belongs to the FGF protein family, which can promote the proliferation of keratinocytes, hair follicle cells, mucosal epithelial cells, etc., so it is also named FGF-7. Human KGF is composed of 163 amino acids, which is a basic protein and contains 5 cysteines, forming two pairs of disulfide bonds. In recent years, a subtype of KGF has been discovered, named KGF-2, which consists of 213 amino acids, and its biological function is similar to that of KGF. The human keratinocyte growth factor described in this patent does not include KGF-2. The FGF (Fibroblast Growth Factor) protein family consists of more than ten proteins with similar structures, mainly including bFGF, aFGF, KGF, KGF-2, I...

Claims

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Application Information

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IPC IPC(8): C07K14/475A61K38/18A61P17/02C12N15/79
Inventor 范开胡伟范可夫
Owner CHONGQING FAGEN BIOMEDICAL
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