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 -