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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 저널정보
- 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제19권 제7호
- 발행연도
- 2009.1
- 수록면
- 727 - 733 (7page)
이용수
초록· 키워드
오류제보하기
Heat shock protein 70 kDa (hsp70), a molecular chaperone
involved in folding of nascent proteins, has been studied
for its ability to activate innate and specific immunity.
High purity hsp70 preparation is generally required for
immunization experiments, because endotoxins and other
immunologically active contaminants may affect immune
responses independently of hsp70. We have developed a
novel modification of E. coli-expression medium that enabled
a simple two-step production and purification method for
endotoxin-free recombinant hsp70. During Ni-NTA-based
affinity purification of hsp70, a contaminating protein
from host E. coli cells, L-glutamine-D-fructose-6-phosphate
aminotransferase (GFAT), was identified. By testing various
compounds, supplementation of growth medium with a
GFAT metabolite, N-acetylglucosamine, was found to reduce
GFAT expression and increase the total hsp70 yield five
times. The new protocol is based on column purification
of His-tagged hsp70 protein produced by E. coli with the
modified medium, followed by endotoxin removal by Triton
X-114 extraction. This approach yielded hsp70 with high
purity and minimal endotoxin contamination, making the
final product acceptable for immunization experiments.
In summary, a simple modification of growth medium
allowed production of recombinant mouse hsp70 in high
yield and purity, thus compatible with immunological studies.
This protocol may be useful for production of other Histagged
proteins expressed in E. coli.
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참고문헌 (24)
참고문헌 신청
Aida, Y. / 1990 / Removal of endotoxin from protein solutions by phase separation using Triton X-114 / J. Immunol. Methods 132 : 191 ~ 195
Bausinger, H. / 2002 / Endotoxin-free heat-shock protein 70 fails to induce APC activation / Eur. J. Immunol 32 : 3708 ~ 3713
Baykov, A. A. / 1988 / A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay / Anal. Biochem 171 : 266 ~ 2
Benaroudj, N. / 1994 / Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70 / Eur. J. Biochem 221 : 121 ~ 128
Geladopoulos, T. P. / 1991 / A malachite green colorimetric assay for protein phosphatase activity / Anal. Biochem 192 : 112 ~ 116
Bausinger, H. / 2002 / Endotoxin-free heat-shock protein 70 fails to induce APC activation / Eur. J. Immunol 32 : 3708 ~ 3713
Baykov, A. A. / 1988 / A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay / Anal. Biochem 171 : 266 ~ 2
Benaroudj, N. / 1994 / Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70 / Eur. J. Biochem 221 : 121 ~ 128
Geladopoulos, T. P. / 1991 / A malachite green colorimetric assay for protein phosphatase activity / Anal. Biochem 192 : 112 ~ 116
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