Please use this identifier to cite or link to this item:
Title: Production of high levels of soluble recombinant Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli
Authors: Sim, B.J.
Tan, D.S.H. 
Liu, X.
Sim, T.S. 
Keywords: Cultivation temperature
Isopenicillin n synthase
Streptomyces clavuligerus
Issue Date: 4-Dec-1996
Citation: Sim, B.J., Tan, D.S.H., Liu, X., Sim, T.S. (1996-12-04). Production of high levels of soluble recombinant Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli. Journal of Molecular Catalysis B: Enzymatic 2 (2-3) : 71-83. ScholarBank@NUS Repository.
Abstract: Streptomyces clavuligerus isopenicillin N synthase (scIPNS) gene expression under the control of T7- and trc-promoters in pET24d and pTrc99A vectors respectively in Escherichia coli was found to be affected by temperature. Although the scIPNS protein is mostly aggregated and inactive in the inclusion bodies when made at 37°C, soluble enzyme is synthesized at 25-28°C. Studies conducted demonstrated that the promoter, as well as the E. coli strains used play critical roles in determining the level of soluble scIPNS made. It is also apparent from computational analysis that the protein structure (perhaps influenced by hydrophobic residues at strategic positions) may also affect the solubility of the expressed scIPNS. However, after genetic manipulation (or under appropriate conditions), overproduction of the scIPNS protein by the T7-promoter to a level of ≈ 29% of total soluble protein in E. coli BL21(DE3) grown at 25°C was achieved and the recombinant enzyme was found to retain activity. It was also observed that soluble scIPNS expressed at 25°C was converted to the insoluble form after incubation in vitro at 37°C, whereas insoluble scIPNS expressed at 37°C remained aggregated regardless of the incubation temperature in vitro. This suggested that the host's milieu affects the solubility (or folding) of the scIPNS expressed.
Source Title: Journal of Molecular Catalysis B: Enzymatic
ISSN: 13811177
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
There are no files associated with this item.

Page view(s)

checked on Apr 19, 2019

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.