Modulation of the tendency towards inclusion body formation of recombinant protein by the addition of glucose in the araBAD promoter system of Escherichia coli.
We attempted to modulate the overall protein expression rate through the addition of a repressor against the araBAD promoter system of Escherichia coli, in which glucose was used as a repressor. Therefore, 0.5% L-arabinose was initially contained as an inducer in culture medium, and either 2% glucose or 2% glycerol was used as a carbon source, and it was found that the expression of recombinant interferon-alpha could be observed at the beginning of the batch culture when glycerol was used as a carbon source. However, when glucose was used, the initiation of recombinant interferon-alpha expression was delayed compared with that when glycerol was used. Furthermore, when the addition of 0.5% glucose was carried out once or twice after 0.5% L-arabinose induction during DO-stat fed-batch culture, the distributions of soluble and insoluble recombinant interferon-alpha were modulated. When glucose was not added after the induction of L-arabinose, all of the expressed recombinant interferon-alpha formed an inclusion body during the later half of culturing. However, when glucose was added after induction, the expressed recombinant interferon-alpha, did not all form an inclusion body, and about half of the total recombinant interferon-alpha was expressed in a soluble form. It was deduced that the addition of glucose after the induction of L-arabinose might lower the cAMP level, and thus, CAP (catabolite activator protein) might not be activated. The transcription rate of recombinant interferon-alpha in the araBAD promoter system might be delayed by the partial repression. This inhibition of the transcription rate probably resulted in more soluble interferon-alpha expression caused by the reduction of the protein synthesis rate.
Lee
You-Jin
YJ
Division of Food and Biotechnology, Chungju National University, Chungju 380-702, Korea.
Jung
Kyung-Hwan
KH
D004926:Escherichia coli / Q000235:genetics*; D005947:Glucose / Q000494:pharmacology*; D002479:Inclusion Bodies / Q000378:metabolism*; D007370:Interferon Type I / Q000096:biosynthesis*; D016898:Interferon-alpha / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins
D007370:Interferon Type I; D016898:Interferon-alpha; D011994:Recombinant Proteins; D005947:Glucose