Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium.
A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His(6)-tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry.
Biedendieck
Rebekka
R
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
Yang
Yang
Y
Deckwer
Wolf-Dieter
WD
Malten
Marco
M
Jahn
Dieter
D
D001410:Bacillus megaterium / Q000737:chemistry / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease; D015951:Factor Xa