Details
| Original language | English |
|---|---|
| Pages (from-to) | 194-202 |
| Number of pages | 9 |
| Journal | Journal of biotechnology |
| Volume | 140 |
| Issue number | 3-4 |
| Publication status | Published - 29 Jan 2009 |
Abstract
Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.
Keywords
- Affinity purification, Escherichia coli, Extracellular production, Maltose binding protein, Recombinant protein, Secretory expression
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Journal of biotechnology, Vol. 140, No. 3-4, 29.01.2009, p. 194-202.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein
AU - Sommer, Benjamin
AU - Friehs, Karl
AU - Flaschel, Erwin
AU - Reck, Michael
AU - Stahl, Frank
AU - Scheper, Thomas
N1 - Funding information: This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG; project FR 837/3). Their support is gratefully acknowledged. LppBRP DNA was a kind gift of B. Oudega from the Vrije Universiteit Amsterdam, The Netherlands. Plasmids p55 and p286 were constructed and donated by G. Miksch. The authors appreciate the generous provision of these plasmids.
PY - 2009/1/29
Y1 - 2009/1/29
N2 - Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.
AB - Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.
KW - Affinity purification
KW - Escherichia coli
KW - Extracellular production
KW - Maltose binding protein
KW - Recombinant protein
KW - Secretory expression
UR - http://www.scopus.com/inward/record.url?scp=62849120953&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2009.01.010
DO - 10.1016/j.jbiotec.2009.01.010
M3 - Article
C2 - 19428714
AN - SCOPUS:62849120953
VL - 140
SP - 194
EP - 202
JO - Journal of biotechnology
JF - Journal of biotechnology
SN - 0168-1656
IS - 3-4
ER -