Novel expression systems for recombinant protein production at low temperatures Maria Giuliani 1 , Ermenegilda Parrilli 1,2 , Maria Luisa Tutino 1,2 , Giovanni Sannia 1 and Gennaro Marino 1,2 1 Department of Organic Chemistry and Biochemistry, University of Naples “Federico II” Complesso Universitario M.S. Angelo via Cinthia 4, 80126, Napoli Italia 2 Faculty of Biotechnological sciences, University of Naples “Federico II” One of the main limitations experienced while producing proteins in conventional bacterial mesophilic systems is the need to operate at their optimal growth temperature (usually 37 ºC) for the production process. Since temperature has a general negative impact on protein folding due to the strong temperature dependence of hydrophobic interactions that mainly drive the aggregation reaction, the production of recombinant proteins at low temperatures represents an exciting model to improve the quality of the products. Recombinant protein production in psychrophilic bacteria, i.e. at temperature as low as 4°C, may minimise undesired hydrophobic interactions during protein folding, desirably resulting in enhancing the yield of soluble and correctly folded products. In this context, a few cold adapted species are under early but intense exploration as cold cell factories, among them, Pseudoalteromonas haloplanktis being a representative example. The efficiency of cold-adapted expression systems was tested by fully soluble and biologically competent production of several thermal-labile and aggregation-prone products in PhTAC125 such as the mature human nerve growth factor and a yeast α- glucosidase Furthermore, with respect to E. coli, PhTAC125 is extremely efficient in secreting proteins in the culture medium. By the use of a psychrophilic α-amylase as secretion carrier for the extra-cellular targeting of recombinant proteins an efficient gene-expression system was set up. Observed efficiency of the cold-adapted system (secretion yield was always above 80%) placed it amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. 1. Introduction The number of candidate proteins to be used as biopharmaceuticals or in industrial processes is rapidly increasing in recent years (Pavolu et al., 2005). However, efficient expression of genes in homologous/heterologous expression systems and rapid purification steps are actually major bottlenecks. In fact, although many recombinant proteins have been successfully produced by common prokaryotic (Escherichia coli) and eukaryotic (yeasts and CHO cells) hosts, these conventional systems have often proved to be unproductive due to the special properties of the protein to be produced. Indeed, beside the obvious impossibility of achieving a large scale production of thermally labile proteins at the normal E. coli growth temperature, degradation of the product by the host proteases and the incorrect folding of the nascent polypeptides, resulting in the protein aggregation and accumulation as insoluble inclusion bodies, are sometimes observed (Speed et al., 1996). To overcome the above mentioned limits of E. coli as host for recombinant protein production, a rational experimental approach has