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Nucleoside diphosphokinase A oxidation-reduction isomer

A technology of nucleoside diphosphate kinase and isomer, applied in the field of genetic engineering, can solve the problems of loss, loss of NDPK activity, loss of protein phosphotransferase activity, etc.

Active Publication Date: 2007-10-31
广州少伯控股集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Freije JM et al. conducted site-directed mutation studies on the Nm23-H1 gene, showing that the serine at position 120 was mutated to glycine (S120G) and the proline at position 96 was mutated to serine (P96S), which retained NDPK activity but lost histidine Mutations lacking motility suppressive capacity upon transfection are deficient in histidine -dependent protein phosphortransferase pathways in vitro.J Biol-Chem, 1997); Kim YI and others mutated the 118-position histidine to phenylalanine (H118F), although it did not affect the function of Nm23-H1 gene to suppress tumor metastasis, but Loss of NDPK activity (Kim YI, Park S, Jeoung DI, et al. Point mutations affecting theoligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells. Biochem Biophys ResCommun, 2003)
[0012] All that can be retrieved so far are reports on site-directed mutations of key active residues of NDPK-A. The purpose of these studies is to clarify the decisive role of a certain site on a specific activity of NDPK-A, so the isomers are all or More or less loss of certain activity of NDPK

Method used

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  • Nucleoside diphosphokinase A oxidation-reduction isomer
  • Nucleoside diphosphokinase A oxidation-reduction isomer
  • Nucleoside diphosphokinase A oxidation-reduction isomer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of mutant Nm23-H1 gene by PCR method

[0043] 1. Purpose

[0044] In the previous work, we found (Xiong Sheng, Qian Chuiwen, Wang Yifei, et al. Research on the Isomerization and Mechanism of Nucleoside Diphosphate Kinase A. Chinese Journal of Biochemistry and Molecular Biology. 2005), recombinant human NDPK-A can be Isomerization occurs due to oxidation and reduction, and some physicochemical properties and enzyme activities of isomers are significantly different. These phenomena suggest that the mutation of key residues (Cys) related to redox isomerization of NDPK-A may improve its activity.

[0045] 2. Experimental materials

[0046] The expression plasmid pBV-Nm23H1 containing the wild-type NDPK-A gene was constructed by Hou Yu et al. and kept in our laboratory (Hou Yu et al. Expression of Nm23-H1 / NDPK-A gene in Escherichia coli and purification of its product. Chinese Journal of Biochemistry and Molecular Biology, 1998), E.coli DH5α was pu...

Embodiment 2

[0061] Embodiment 2: Construction of NDPK-A C4S engineering bacteria

[0062] 1. Purpose

[0063] Construction of genetically engineered bacteria capable of highly expressing NDPK-A C4S mutant protein.

[0064] 2. Experimental materials

[0065] Recombinant plasmid pUC-Nm23H1 C4S containing NDPK-A C4S gene (constructed by Example 1), E.coliDH5α was purchased from Promega Company, expression vector pBV220 (Zhang Zhiqing et al. containing P_RP_L promoter, prokaryotic high-efficiency expression vector, and its application virus Acta 1990).

[0066] 3. Experimental method

[0067] Preparation of exogenous gene fragments: E.coli DH[pUC-Nm23H1 C4S] single colony inoculates 5mL LB culture medium, cultivates overnight at 37°C and 220rpm, extracts plasmid DNA by mini-extraction method, double enzyme digestion with EcoR I and BamH I, enzyme digestion The product was subjected to agarose gel electrophoresis, and small fragments were recovered from the gel, and stored at -20°C for fut...

Embodiment 3

[0075] Example 3: Expression and purification of NDPK-A isoforms

[0076] 1. Purpose

[0077] Prepare NDPK-A C4S isoform protein to provide materials for subsequent experiments such as activity determination.

[0078] 2. Experimental materials

[0079] The engineering bacteria E.coli DH[pBV-Nm23-C4S] was preserved by the inventor's laboratory, and the construction method was listed in Example 2; the chromatography matrix DEAE-sepharose Fast Flow was purchased from Amersham Bioscience Company; Cibacron Blue3GA Sepharose CL-4B Purchased from Millipore Company;

[0080] Purification buffer and its formula: Buffer A: 20mmol / L Tris-HCl, 1mmol / L EDTA, 2mmol / LMgCl 2 , 1mmol / L DTT, pH7.5; Buffer B: Buffer A containing 0.05mol / L NaCl; Buffer C: BufferA containing 0.1mol / L NaCl; Buffer D: Buffer C containing 1.5mmol / L NaCl.

[0081] 3. Experimental method

[0082] 3.1 Shake flask fermentation and sample pretreatment

[0083] Inoculate 5mL LB medium with engineering bacteria DH[pBV...

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Abstract

The invention discloses a method to get nucleic diphosphokinase A (NDPK-A) oxidation-reduction isomer through gene engineering in genetic engineering domain, which is characterized by the following: producing a amino acid abrupt change in NDPK-A; generating the oxidation-reduction isomer; abrupt-changing forth position cysteine to serine; getting amino acid abrupt change; setting the amino acid sequence as SEQ ID NO. 2. This isomer possesses stronger activity, which can increase activity of phosphate group transferase of NDPK and DNase (inhibit cancer).

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a redox isomer of nucleoside diphosphate kinase A (NDPK-A) obtained through genetic engineering. Background technique [0002] The research history of nucleoside diphosphate kinase (NDPK) can be traced back 50 years ago (Berg P, Joklik WK. Transphosphorylation between nucleoside polyphosphates. Nature, 1953). As a housekeeping enzyme, the primary function of NDPK is to pass Catalyzes the phosphate transfer reaction between NDP and NTP to maintain the concentration of NTP in the cell. Recent studies have found that NDPK can regulate cell proliferation, differentiation, development (Rosengard AM, Krutzsch HC, Shearn A, et al. Reduced Nm23 / Awd protein intumour metastasis and aberrant Drosophila development. Nature, 1989) and apoptosis (Fan Z , Beresford PJ, Oh DY, et al.Tumor suppressor NM23-H1 is a granulzyme A-activated DNaseduring CTL-mediated apoptosis, and the nucleosome ass...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54A61K38/45A61P31/12A61P35/00
Inventor 熊盛王一飞郭朝万
Owner 广州少伯控股集团有限公司
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