Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium

• Published in: Biotechnology and bioengineering 2007-02, Vol.96 (3), p.525-537
• Main Authors: Biedendieck, Rebekka, Yang, Yang, Deckwer, Wolf-Dieter, Malten, Marco, Jahn, Dieter
• Format: Article
• Language: English
• Subjects: affinity tags; Bacillus megaterium; Bacillus megaterium - chemistry; Bacillus megaterium - genetics; Biological and medical sciences; Biotechnology; Chromatography, Affinity - methods; Cloning, Molecular; Cytoplasm - chemistry; Cytoplasm - genetics; Endopeptidases - chemistry; Endopeptidases - genetics; Factor Xa - chemistry; Factor Xa - genetics; fed batch cultivation; Fundamental and applied biological sciences. Psychology; Genetic Vectors; green fluorescent protein; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - isolation & purification; Histidine - chemistry; Histidine - genetics; Plasmids - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; recombinant protein production; Tobacco etch virus
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.21145

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 His6‐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. Biotechnol. Bioeng. 2007;96: 525–537. © 2006 Wiley Periodicals, Inc.