A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium.
Gene expression systems based on the RNA polymerase of the bacteriophage T7 are often the ultimate choice for the high level production of recombinant proteins. During the last decade, the Gram-positive bacterium Bacillus megaterium was established as a useful host for the intra- and extracellular production of heterologous proteins. In this paper, we report on the development of a T7 RNA polymerase-dependent expression system for B. megaterium. The system was evaluated for cytosolic and secretory protein production with green fluorescent protein (GFP) from Aequoria victoria as intracellular and Lactobacillus reuteri levansucrase as extracellular model protein. GFP accumulated rapidly at high levels up to 50 mg/l shake flask culture intracellularly after induction of T7 RNA polymerase gene expression. The addition of rifampicin for the inhibition of B. megaterium RNA polymerase led to an increased stability of GFP. L. reuteri levansucrase was also successfully produced and secreted (up to 20 U/l) into the culture supernatant. However, parallel intracellular accumulation of the protein indicated limitations affiliated with the Sec-dependent protein translocation process.
Gamer
Martin
M
Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Fröde
David
D
Biedendieck
Rebekka
R
Stammen
Simon
S
Jahn
Dieter
D
D001410:Bacillus megaterium / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D006602:Hexosyltransferases; C021513:levansucrase; D012321:DNA-Directed RNA Polymerases