title: Regulated recombinant protein production in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.
abstract: This review reports results from our laboratory on the development of an effective inducible expression system for the homologous/heterologous protein production in cold-adapted bacteria. Recently, we isolated and characterized a regulative genomic region from Pseudoalteromonas haloplanktis TAC125; in particular, a two-component regulatory system was identified. It is involved in the transcriptional regulation of the gene coding for an outer membrane porin (PSHAb0363) that is strongly induced by the presence of L: -malate in the growth medium.We used the regulative region comprising the two-component system located upstream the PSHAb0363 gene to construct an inducible expression vector - named pUCRP - under the control of L: -malate. Performances of the inducible system were tested using the psychrophilic β-galactosidase from P. haloplanktis TAE79 as model enzyme to be produced. Our results demonstrate that the recombinant cold-adapted enzyme is produced in P. haloplanktis TAC125 in good yields and in a completely soluble and catalytically competent form. Moreover, an evaluation of optimal induction conditions for protein production was also carried out in two consecutive steps: (1) definition of the optimal cellular growth phase in which the gene expression has to be induced; (2) definition of the optimal inducer concentration that has to be added in the growth medium.
authors.affiliation: Department of Organic Chemistry and Biochemistry, Università degli studi di Napoli Federico II, Naples, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Rippa
authors.forename: Rosanna
authors.initials: R
authors.lastname: Papa
authors.forename: Maria
authors.initials: M
authors.lastname: Giuliani
authors.forename: Cinzia
authors.initials: C
authors.lastname: Pezzella
authors.forename: Ermenegilda
authors.initials: E
authors.lastname: Parrilli
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Tutino
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
authors.forename: Angela
authors.initials: A
authors.lastname: Duilio
mesh_terms: D000220:Adaptation, Biological / Q000502:physiology*; D000864:Antarctic Regions; D003080:Cold Temperature; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics; D008293:Malates; D009841:Oligonucleotides / Q000235:genetics; D018272:Porins / Q000378:metabolism; D031161:Pseudoalteromonas / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D003470:Culture Media; D008293:Malates; D009841:Oligonucleotides; D018272:Porins; D011994:Recombinant Proteins; C030298:malic acid; D001616:beta-Galactosidase