Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein.
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 alpha-1,4-glucans.
Department of Fermentation Engineering, Faculty of Technology, Bielefeld University, POB 100 131, D-33501 Bielefeld, Germany.
Benjamin
B
Sommer
Karl
K
Friehs
Erwin
E
Flaschel
Michael
M
Reck
Frank
F
Stahl
Thomas
T
Scheper
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005822:Genetic Vectors; D005936:Glucans / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D058619:Maltose-Binding Proteins; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005936:Glucans; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C040088:1,4-glucan; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease