Hart, Roger A. (1991) Characterization of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06272007-152616
The process of inclusion body (IB) formation in the gram-negative bacterium Escherichia coli (E. coli) was investigated. The homodimeric hemeprotein Vitreoscilla hemoglobin (VHb) from the gram-negative bacterium Vitreoscilla was taken as the model protein.
Expression of VHb under control of its native promoter from a pUC19-derived plasmid in strain JM101 lead to high-level accumulation of VHb in both soluble and insoluble forms. The soluble form was purified by sequential two-phase extraction techniques and used as a basis for analyzing the insoluble form. The amino acid content and N-terminal sequence of purified soluble VHb is consistent with that of VHb purified from Vitreoscilla. Soluble and insoluble VHb exhibit identical migration properties during denaturing two-dimensional electrophoresis.
The protein composition of VHb inclusion bodies was analyzed by one-dimensional and two-dimensional electrophoresis techniques. Results indicate the presence of two types of cytoplasmic aggregates of differing morphology in single bacterial cells. These aggregates also differ in their relative content of VHb, pre-[beta]-lactamase, and the cytoplasmic protein elongation factor Tu and are separable by differential centrifugation.
Conformational properties of soluble and insoluble VHb were studied by electron paramagnetic resonance spectroscopy. Purified soluble VHb exhibits three high-spin resonances in the vicinity of g 6 from two heme centers. One center is axial (g 6.00). The other is rhombic (g 5.50 and 6.39). Inclusion body isolates containing insoluble VHb exhibit a single resonance (g 5.98) which is also present in control cell debris. Iron quantitation demonstrates that inclusion body VHb uniformly lacks heme. Titration of IB fractions with monomeric ferrous heme followed by difference absorption spectroscopy suggests that some inclusion body VHb is competent for heme binding.
A series of perturbation-response experiments was conducted to determine what cellular processes influence VHb IB formation. Results show that VHb inclusion body formation is highly influenced by the expression plasmid construction and the heme biosynthetic capacity. The level of induction and accumulation appear less important than the general metabolic state of the culture. Temperature and chaperone protein levels have little effect. Efforts to reduce inclusion body formation through genetic amplification of ALA synthase and ALA dehydratase levels were unsuccessful, presumably due to regulation. Results suggest a heme biosynthetic limitation is involved in VHb in vivo insolubilization.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||23 May 1991|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||20 Jul 2007|
|Last Modified:||26 Dec 2012 02:54|
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