Expression, Single-Step Purification and Matrix-Assisted
Refolding of a Maize Cytokinin Glucoside-Specific
beta-Glucosidase

J 1999

Expression, Single-Step Purification and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific beta-Glucosidase

ZOUHAR, Jan; Elizabeth NANAK a Břetislav BRZOBOHATÝ

Základní údaje

Originální název

Expression, Single-Step Purification and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific beta-Glucosidase

Autoři

ZOUHAR, Jan; Elizabeth NANAK a Břetislav BRZOBOHATÝ

Vydání

Protein Expression and Purification, San Diego, California, Academic Press, 1999, 1046-5928

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

Genetika a molekulární biologie

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Impakt faktor

Impact factor: 1.416

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/99:00001149

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

beta-glucosidase; immobilized metal affinity chromatography; perfusion matrix; refolding

Štítky

beta-glucosidase, immobilized metal affinity chromatography, perfusion matrix, refolding

Změněno: 7. 12. 2001 16:29, prof. RNDr. Břetislav Brzobohatý, CSc.

Anotace

V originále

Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)6Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)6Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after refolding process was confirmed by Km determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.

Návaznosti

MSM 143100008, záměr

Název: Genomy a jejich funkce

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Genomy a jejich funkce

VS96096, projekt VaV

Název: Laboratoř molekulární fyziologie rostlin

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Laboratoř molekulární fyziologie rostlin

Citovat

ZOUHAR, Jan; Elizabeth NANAK a Břetislav BRZOBOHATÝ. Expression, Single-Step Purification and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific beta-Glucosidase. Protein Expression and Purification. San Diego, California: Academic Press, 1999, roč. 1999, č. 17, s. 153-162. ISSN 1046-5928.

@article{209591,
   author = {Zouhar, Jan and Nanak, Elizabeth and Brzobohatý, Břetislav},
   article_location = {San Diego, California},
   article_number = {17},
   keywords = {beta-glucosidase; immobilized metal affinity chromatography; perfusion matrix; refolding},
   language = {eng},
   issn = {1046-5928},
   journal = {Protein Expression and Purification},
   title = {Expression, Single-Step Purification and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific beta-Glucosidase},
   volume = {1999},
   year = {1999}
}

TY  - JOUR
ID  - 209591
AU  - Zouhar, Jan - Nanak, Elizabeth - Brzobohatý, Břetislav
PY  - 1999
TI  - Expression, Single-Step Purification and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific beta-Glucosidase
JF  - Protein Expression and Purification
VL  - 1999
IS  - 17
SP  - 153
EP  - 153
PB  - Academic Press
SN  - 10465928
KW  - beta-glucosidase
KW  - immobilized metal affinity chromatography
KW  - perfusion matrix
KW  - refolding
N2  - Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)6Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)6Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after refolding process was confirmed by Km determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.
ER  -

ZOUHAR, Jan; Elizabeth NANAK a Břetislav BRZOBOHATÝ. Expression, Single-Step Purification and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific beta-Glucosidase. \textit{Protein Expression and Purification}. San Diego, California: Academic Press, 1999, roč.~1999, č.~17, s.~153-162. ISSN~1046-5928.

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