Rippa Valentina, Papa Rosanna, Giuliani Maria, Pezzella Cinzia, Parrilli Ermenegilda, Tutino Maria Luisa, Marino Gennaro, Duilio Angela
Department of Organic Chemistry and Biochemistry, Università degli studi di Napoli Federico II, Naples, Italy.
Methods Mol Biol. 2012;824:203-18. doi: 10.1007/978-1-61779-433-9_10.
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.
本综述报告了我们实验室在开发一种有效的诱导表达系统方面的成果，该系统用于在冷适应细菌中生产同源/异源蛋白质。最近，我们从嗜盐栖冷假交替单胞菌TAC125中分离并鉴定了一个调控基因组区域；特别地，鉴定出了一个双组分调控系统。它参与编码外膜孔蛋白（PSHAb0363）的基因的转录调控，该基因在生长培养基中L-苹果酸存在时被强烈诱导。我们使用包含位于PSHAb0363基因上游的双组分系统的调控区域，构建了一个在L-苹果酸控制下的诱导表达载体——命名为pUCRP。使用来自嗜盐栖冷假交替单胞菌TAE79的嗜冷β-半乳糖苷酶作为要生产的模型酶，测试了诱导系统的性能。我们的结果表明，重组冷适应酶在嗜盐栖冷假交替单胞菌TAC125中以高产率产生，并且以完全可溶且具有催化活性的形式存在。此外，还分两个连续步骤对蛋白质生产的最佳诱导条件进行了评估：（1）确定必须诱导基因表达的最佳细胞生长阶段；（2）确定必须添加到生长培养基中的最佳诱导剂浓度。
