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Recombinant beta-galactosidase derived from Streptococcus pneumaniae

a technology of streptococcus pneumaniae and beta-galactosidase, which is applied in the direction of peptide/protein ingredients, drug compositions, enzymology, etc., can solve the problems of inability to commercialize drugs able to inhibit the metastasis of cancer cells, problems that may still exist, and the effect of preventing cancer cell growth

Inactive Publication Date: 2008-10-09
KOREA RES INST OF BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021]It is still another object of the present invention to provide a method for inhibiting cancer cell growth using the protein.

Problems solved by technology

However, even though the primary cancer has been eradicated, some problems may still exist.
That is, cancer may be called as a malignant tumor because of its metastatic ability, and in many cases, metastatic cancer is more likely to cause death.
It cannot be said yet that a method for inhibiting the metastasis of cancer cells has been established, and a medicine having the effects of inhibiting the metastasis of cancer cells has not yet been commercially available.
However, BgaC proteins, which are galactosidases capable of cleaving the galactose linked by beta1,3-linkage, have not been well known, until now.

Method used

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  • Recombinant beta-galactosidase derived from Streptococcus pneumaniae
  • Recombinant beta-galactosidase derived from Streptococcus pneumaniae
  • Recombinant beta-galactosidase derived from Streptococcus pneumaniae

Examples

Experimental program
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example 1

Amplification of bgaC Gene from Streptococcus pneumoniae

[0054]A Streptococcus pneumoniae chromosome was isolated from Streptococcus pneumoniae (ATCC BBA-255D) using a known phenol extraction (Ushiro et al., J Dent Res 70: 1422-1426, 1991). Polymerase chain reaction (PCR) was performed using the extracted chromosomal DNA as a template, and a pair of primer, bgaC-N(cgctagCATGACACGATTTGAGATACGAG) and bgaC-C(ggaagcttTCATAAGTTTTCCCCCTTTATATG), at which NheI and HindIII enzyme restriction sites were artificially inserted, so as to prepare a DNA fragment containing bgaC with a size of 1.8 kb (see FIG. 1A).

[0055]The base sequence of the prepared DNA fragment was translated. As a result, it was found that the Streptococcus pneumoniae bgaC gene encodes an intracellular protein consisting of 595 amino acids. From the result of translating into the amino acid sequence, it was found that the Streptococcus pneumoniae BgaC protein has 29.8%, 54.3%, 65.4%, and 46.7% homologies and 40.8%, 58.1%, 41...

example 2

Large-scale Expression, Isolation, and Purification of BgaC Protein

[0056]A recombinant vector pET28a (Novagen) was cleaved with NheI and HindIII restriction enzymes, and the PCR product, which is treated with the same enzymes and amplified in Example 1, was introduced into the vector (FIG. 1B). The prepared recombinant vector was transformed into E. coli BL21 (DE3) strain. The transformed E. coli was precultured in 5 ml of LB liquid medium (1% Bacto Tripton, 1% Sodium chloride, 0.5% Yeast extract) at 37° C. for 16 hours. The precultured medium was inoculated into fresh LB medium at a dilution of 1:100, and cultured at 37° C. When the absorbance of the medium was 0.4 to 0.6 at 600 nm, IPTG (isopropyl thiogalactoside) was added thereto to be a final concentration of 1 mM. Thus, the BgaC protein expression was induced and the medium was cultured at 18° C. for 24 hours. The cultured E. coli cells were centrifuged, recovered, and then disrupted by sonication. The disrupted cells were cen...

example 3

Galactosidase Activity Test for BgaC Protein

[0057]The basic activity of the BgaC protein was confirmed using ONPG (o-nitrophenyl-D-galactopyranoside) and PNPG (p-nitrophenyl-D-galactopyranoside) as a substrate. 2.2 mg of BgaC protein was mixed with a reaction mixture [90 mM sodium phosphate (NaPO4) (pH 6.5), 10 mM magnesium chloride (MgCl2), 45 mM beta-mercaptoethanol, 0.3 mM ONPG or PNPG] to be 300 μl, and reacted at 30° C. for 30 minutes. Then, its absorbance was measured at 420 nm to determine the amount of ONP (o-nitrophenol) or PNP (p-nitrophenol) produced. The activity of BgaC protein (Unit) was defined as ability to convert 1 nmole of ONPG or PNPG into ONP or PNP at 30° C. for 1 minute. The maximum reaction rate of the enzyme is 2.6 times higher in the case of using PNPG than in the case of using ONPG as the substrate. Further, the substrate affinity of the enzyme is 3.5 times stronger for PNPG than for ONPG (see FIG. 3A). In accordance with the Example, it was found that the...

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Abstract

The present invention relates to a novel beta-galactosidase derived from Streptococcus pneumoniae, a BgaC protein exhibiting the enzyme activity, and a method for using the same. The protein can be used in the modification and analysis of sugar chain and used as an anti-cancer agent.

Description

[0001]This application claims priority to Korean Patent Application Ser. No. KR 10-2006-0057140, filed Jun. 23, 2006, which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to an enzyme, BgaC protein having beta-galactosidase (EC 3.2.1.23) activity derived from Streptococcus pneumoniae, and a method for using the same.[0004]2. Description of the Related Art[0005]Beta-galactosidase is a cleavage enzyme that belongs to the family 35 of glycohydrolases, and that is found in plants and animals, as well as in a wide variety of microorganisms such as yeasts, fungi, bacteria, and archaea. Beta-galactosidase hydrolyzes lactose and its structurally related compounds, and additionally catalyzes transgalactosylation reaction of various beta-D-galactopyranosides including lactose. The hydrolase and transferase activity of beta-galactosidase are useful for industrial applications (Nkayama and Amachi, 1999; Hung and ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/47C12N9/38C12N15/56C12N15/63C12Q1/34A61P35/00
CPCA61K38/00C12Y302/01023C12N9/2471A61P35/00C12N9/2468C12N9/00C12N15/10
Inventor KANG, HYUN AHJEONG, JAE KAPKWON, OHSUKOH, DOO-BYOUNGKIM, SEONGHUN
Owner KOREA RES INST OF BIOSCI & BIOTECH
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