Affiliation
a Immanuel Kant Baltic Federal University
b Kemerovo State University
Copyright ©Babich et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0. (
http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Abstract
The pal gene coding for L-phenylalanine ammonia-lyase of Rhodosporidium toruloides (GenBank entry no. X12702.1) with optimized sequence was cloned into an expressing vector pET28a. Three parameters of expression (inductor type, duration, and temperature of induction) were optimized, which resulted in a strain producing recombinant L-phenylalanine ammonia-lyase with the maximal productivity, that is, 35 ± 1% to total cell protein, upon utilization of 0.2% lactose (according to Studier) induction during 18 h at 37°C. The recombinant L-phenylalanine ammonia-lyase was found to be insoluble by 99%. Solubility of the protein did not improve upon utilization of 1 mM IPTG as an inductor instead of 0.2% lactose, or upon bacterium cultivation at various temperatures, that is 25°C and 37°C.
Keywords
L-phenylalanine ammonia-lyase,
cloning,
expression,
recombinant protein,
induction,
L-phenylalanine,
phenylketonuria
REFERENCES
- Sarkissian, C.N., and Gamez, A., Phenylalanine ammonia lyase, enzyme substitution therapy for phenylketonuria, where are we now? Mol. Genet. Metab., 2005, vol. 86, pp. S22–26.
- Sarkissian, C.N., Shao, Z., Blain, F., Peevers, R., Su, H., Heft, R., Chang, T.M.S., and Scriver, C.R., A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase, Proc. Natl. Acad. Sci. U.S.A., 1999. vol. 96, no. 5, pp. 2339–2344.
- Evans, C.T., Hanna, K., Payne, C., Conrad, D., and Misawa, M., Biotransformation of trans-cinnamic acid to L-phenylalanine: Optimization of reaction conditions using whole yeast cells, Enzyme Microb. Technol., 1987, vol. 9, pp. 417–421.
- Baneyx, F., Recombinant protein expression in Escherichia coli, Curr. Opin. Biotechnol., 1999, vol., 10, pp. 411–421.
- Hannig, G., and Makrides, S.C., Strategies for optimizing heterologous protein expression in Escherichia coli, Trends in Biotechnology, 1998, vol. 16, pp. 54–60.
- Beckwith, J., The operon: An historical account in Escherichia Coli and Salmonella Typhimurium: Cellular and Molecular Biology, Neidhardt, F.C., Ed., Washington, D.C.: American Society for Microbiology, 1987, pp. 1439–1452.
- Studier, F.W., Protein production by auto-induction in high density shaking cultures, Protein Expr. Purif., 2005, vol. 41, pp. 207–234.
- Kido, M., Yamanaka, K., Mitani, T., Niki, H., Ogura, T., and Hiraga, S., RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli, J. Bacteriol., 1996, vol. 178, pp. 3917–3925.
- Lopez, P.J., Marchand, I., Joyce, S.A., and Dreyfus, M., The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo, Mol. Microbiol., 1999, vol. 33, pp. 188–199.
- Grossman, T.H., Kawasaki, E.S., Punreddy, S.R., and Osburne, M.S., Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability, Gene, 1998, vol. 209, pp. 95–103.
- Inada, T., Kimata, K., and Aiba, H., Mechanism responsible for glucose-lactose diauxie in Escherichia coli: challenge to the cAMP model, Genes Cells, 1996, vol. 1, pp. 293–301.
- Kimata, K., Takahashi, H., Inada, T., Postma, P., and Aiba, H., cAMP receptor protein-cAMP plays a crucial role in glucose-lactose diauxie by activating the major glucose transporter gene in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A., 1997, vol. 94, pp. 12914–12919.
- Studier, F.W., and Moffatt, B.A., Use of bacteriophage T7 RNA-polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol., 1986, vol. 189, pp. 113–130.
