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- Open Access
Production and purification of high molecular weight oligomers of Yersinia pestis F1 capsular antigen released by high cell density culture of recombinant Escherichia coli cells carrying the caf1 operon
© Holtzman et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Size Exclusion Chromatography
- High Cell Density
- Yersinia Pestis
- High Cell Density Culture
Yersinia pestis fraction 1 antigen (F1) is the major component of the pathogen capsule. F1, a 15.5 kDa monomer that forms high molecular oligomers (>1000 kDa), is a highly protective antigen and is considered as a key constituent of a subunit anti-plague vaccine.
F1 antigen is encoded on the caf1 operon together with a transcriptional regulator (caf1R), a chaperone (caf1M) and an usher protein (caf1A) and its expression is induced by a temperature shift to 37°C. Production and purification of F1 protein from Y. pestis cells is a tedious and time consuming procedure. Use of recombinant Escherichia coli for the production of F1 was reported [1, 3].
Here, we describe an efficient procedure for production and purification of F1 released from the cell surface of E. coli cells, carrying the caf1 operon, grown to a high cell density.
The caf1 operon, derived from Y. pestis Kimberley53 virulent strain , was cloned into the medium copy-number plasmid pBR322-Kanamycin (to yield pBRK-F1), and the expression plasmid was introduced into Escherichia coli MC1060. Under expression conditions the cells were found to produce high levels of F1 as a capsule (Y.L., E.M., S.C., Y.F., unpublished data).
Further purification steps were as follows: (a) 33% saturation ammonium sulfate precipitation, (b) size exclusion chromatography and (c) endotoxin removal resulting in at least 3-log reduction of endotoxin content, to ~200 EU/ml. The highly purified F1 antigen (>90%) consisted of high molecular oligomers with final recovery yield of 14% (125 mg F1/L culture).
1. E. coli cells expressing F1 could be grown to a high cell density in SY medium at 28°C, using a computer-controlled fermentor.
2. Continuous high level expression of F1 protein by recombinant E. coli was achieved during fermentation with no plasmid loss.
3. F1 antigen was massively released from the encapsulated cells at high cell density cultures.
4. High molecular weight oligomers of F1 were purified to >90% following few purification steps. This preparation was used for vaccination experiments in animal models.
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