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- Open Access
Recombinant protein expression system in cold loving microorganisms
© Papa et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Inducible Expression
- Recombinant Protein Expression
- Nascent Polypeptide
- Heterologous Protein Production
- Optimal Induction
Soluble and functional proteins are of high demand in modern biotechnology. Although many recombinant proteins have been successfully obtained from common prokaryotic and eukaryotic hosts, these systems result to be often unproductive due to the peculiar properties of the protein to be produced. Incorrect folding of the nascent polypeptide chains is one of the main problems occurring during heterologous protein production in bacteria. Since formation of inclusion bodies often impairs the recombinant production of valuable proteins, many experimental approaches have been explored to minimize this undesirable effect [1, 2]. Expression of "difficult" proteins has also been carried out by lowering the temperature at the physiological limit allowed for the growth of mesophilic host organisms (between 15 and 18°C for Escherichia coli). Lowering the temperature, in fact, has a pleiotropic effect on the folding process, destabilising the hydrophobic interactions needed for intermediates aggregation . On the basis of the above considerations, a rational alternative to mesophilic organisms is the use of naturally cold-adapted bacteria as hosts for protein production at low temperature (even at around 0°C).
The development of a shuttle genetic system for the transformation of the cold adapted Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 (Ph TAC125) [4, 5] has already been reported. This system has made possible the isolation of constitutive psychrophilic promoters and the construction of cold expression systems for the protein production at low temperatures . The described expression system represented the first example of heterologous protein production based on a true cold-adapted replicon . However, the development of an effective cold expression system with industrial perspectives needs to be finely tuned possibly using ad hoc promoters. Physical separation between bacterial growth phase and expression of the desired proteins, in fact, can not only improve the productivity of the entire system but can also play an important role in the production of proteins toxic for the host cells. These goals can only be achieved by using regulated promoters and efficient induction strategies. Recently, using a proteomic approach and exploiting the information deriving from the genome sequence of Ph TAC125  we isolated and characterized a functionally active two-component system involved in the transcriptional regulation of the gene coding for an outer membrane porin, that is strongly induced by the presence of L-malate in the medium .
In this paper we used the regulative region upstream of the porin gene to construct an inducible expression vector, named pUCRP, that is under the control of L-malate. Performances of the inducible system were tested for both psychrophilic and mesophilic protein production using two "difficult" proteins as model systems. Moreover, an evaluation of optimal induction conditions for protein production was also carried out.
- Mitra A, Chakrabarti KS, Shahul Hameed MS, Srinivas KV, Senthil Kumar G, Sarma SP: High level expression of peptides and proteins using cytochrome b5 as a fusion host. Protein Expr Purif. 2005, 41: 84-97. 10.1016/j.pep.2004.12.025.View ArticleGoogle Scholar
- Luo ZH, Hua ZC: Increased solubility of glutathione S-transferase-P16 (GST-p16) fusion protein by co-expression of chaperones groes and groel in Escherichia coli. Biochem Mol Biol Int. 1998, 46: 471-477.Google Scholar
- Jeon YH, Negishi T, Shirakawa M, Yamazaki T, Fujita N, Ishihama A, Kyogoku Y: Solution structure of the activator contact domain of the RNA polymerase alpha subunit. Science. 1995, 270: 1495-1497.View ArticleGoogle Scholar
- Birolo L, Tutino ML, Fontanella B, Gerday C, Mainolfi K, Pascarella S, Sannia G, Vinci F, Marino G: Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125. Cloning, expression, properties, and molecular modelling. Eur J Biochem. 2000, 267: 2790-2802. 10.1046/j.1432-1327.2000.01299.x.View ArticleGoogle Scholar
- Tutino ML, Duilio A, Parrilli E, Remaut E, Sannia G, Marino G: A novel replication element from an Antarctic plasmid as tool for the expression of proteins at low temperatures. Extremophiles. 2001, 5: 257-264. 10.1007/s007920100203.View ArticleGoogle Scholar
- Duilio A, Madonna S, Tutino ML, Pirozzi M, Sannia G, Marino G: Promoters from a cold-adapted bacterium: definition of a consensus motif and molecular characterization of UP regulative elements. Extremophiles. 2004, 8: 125-132. 10.1007/s00792-003-0371-2.View ArticleGoogle Scholar
- Duilio A, Marino G, Mele A, Sannia G, Tutino ML: Sistema di espressione di proteine ricombinanti a basse temperature. Uff It Brev Marchi RM2003/A000155Google Scholar
- Medigue C, Krin E, Pascal G, Barbe V, Bernsel A, Bertin PN, Cheung F, Cruveiller S, D'Amico S, Duilio A, Fang G, Feller G, Ho C, Mangenot S, Marino G, Nilsson J, Parrilli E, Rocha EP, Rouy Z, Sekowska A, Tutino ML, Vallenet D, von Heijne G, Danchin A: Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome Res. 2005, 10: 1325-1335. 10.1101/gr.4126905. 10.1101/gr.4126905.View ArticleGoogle Scholar
- Papa R, Glagla S, Danchin A, Schweder T, Marino G, Duilio A: Proteomic identification of two-component regulatory system in Pseudoalteromonas haloplanktis TAC125. Extremophiles. 2005, , Google Scholar
- Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C: Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis. Appl Environ Microbiol. 2001, 67: 1529-1535. 10.1128/AEM.67.4.1529-1535.2001.View ArticleGoogle Scholar
- Kopetzki E, Buckel P, Schumacher G: Cloning and characterization of baker's yeast alpha-glucosidase: over-expression in a yeast strain devoid of vacuolar proteinases. Yeast. 1989, 5: 11-24. 10.1002/yea.320050104.View ArticleGoogle Scholar
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