Solubility and proteolysis of the Zb-MalE and Zb-MalE31 proteins during overproduction in Escherichia coli.
From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full-length protein, accumulation of the model fusion proteins Zb-MalE and Zb-MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed-batch cultivation. Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb-MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure. It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a "high" feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (< or = 5 microM), this formation is almost abolished while at a comparatively high concentration (> or = 300 microM) 50% of the total product accumulated was in the form of inclusion bodies.
Sandén
Anna Maria
AM
The Swedish Centre for Bioprocess Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden.
Boström
Maria
M
Markland
Katrin
K
Larsson
Gen
G
D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007544:Isopropyl Thiogalactoside; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012995:Solubility
D002352:Carrier Proteins; D003470:Culture Media; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside