However, it is common that heterologous proteins fail to fold correctly at optimal E. coli growth temperatures, resulting in formation of insoluble aggregates known as inclusion bodies. A possible solution is recombinant protein expression at reduced growth temperatures, increasing the solubility
of aggregation-prone recombinant proteins, but this is accompanied by a reduction in metabolic rate. The use of cold-shock expression systems, such as pCold, allowed high-level expression of soluble proteins in E. coli. Cold-shock expression vectors (named pColdI, II, III, and IV) are plasmids in which protein expression is under the control of the cspA (cold-shock protein A) promoter in a pUC118 background, with the cspA 5′-UTR and the
cpsA 3′end transcription terminator site. All pCold vectors contain the lac operator sequence immediately upstream of the cspA transcription initiation site, allowing the Selleck Trametinib cold-shock Nutlin-3a cost induction of gene expression by simultaneous addition of IPTG and temperature downshift in E. coli (Qing et al., 2004). These vectors have been used for expressing successfully cold-adapted proteins in E. coli, for example the protease from Pseudoalteromonas sp. QI-1 (Xu et al., 2011), β-galactosidase from Arthrobacter spychrolactaphilus (Nakagawa et al., 2007), and lipase from Psychrobacter sp. G (Lin et al., 2010), among others. However, enzyme aggregation and accumulation in inclusion bodies cannot be entirely solved by this approach. Cui et al. (2011) successfully improved the yield of
soluble cold-active lipase in the E. coli cytoplasm by co-expression with molecular chaperones. The biotechnological implication of this finding is clear. The production of recombinant proteins in cold-adapted bacteria such as Pseudoalteromonas circumvents the slowdown in metabolic rate imposed by the temperature downshift in mesophilic bacteria such as E. coli, thus increasing productivity, and probably solubility and stability. In this regard, authors have developed new vectors to produce heterologous proteins at low temperature using Antarctic genetic resources as described below. The occurrence of bacterial plasmids in Antarctic bacterial isolates was early Tangeritin studied by Kobori et al. (1984). They found that 48 of 155 isolates (31%) carried at least one plasmid and concluded that bacterial plasmids are ubiquitous in this environment. These endogenous plasmids could be used for the development of cloning systems, mainly by genetic engineering and for the overproduction of heat-labile proteins. Tutino et al. (2000) reported for the first time the isolation and characterization of a cold-adapted plasmid, named pTAUp, from the Antarctic gram-negative Psychrobacter sp. strain TA144. This plasmid duplicates in vivo by a rolling-circle mechanism, and several functional and structural features of the Rep initiator protein suggest the existence of a novel subfamily of RC replicons (Tutino et al., 2000). Later, Tutino et al. (2001) and Zhao et al.