Tecnologie ricombinanti per la produzione di proteina HSP70 di Arabidopsis thaliana in organismi eterologhi

Abstract

Heat shock proteins (HSPs) are evolutionary highly conserved proteins that are present in all living organisms. They were discovered in 1962 by Ferruccio Ritossa during an heat shock experiment on fruits flies, but their identification and the term “heat shock proteins” have been coined in the 1974 (Ritossa, 1962; Tissieres et al., 1974). HSPs are classified into several families according their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSP (15 to 30 kDa) and they are located in all cellular and subcellular compartments (nucleus, membrane, cytosol, endoplasmic reticulum, mitochondria, lysosome). They are expressed constitutively (cognate proteins or HSC) in all cells but their expression increases (inducible forms or HSP) after several stressful conditions, such as environmental (high temperature, oxidative stress, ultraviolet irradiation, heavy metals), pathological (viral or bacterial infections, inflammatory and malignancies) or physiological stimuli (cellular differentiation, hormones and growth factories (Ellen et al., 2002; Wang et al., 2004). Their overexpression is essential for survival of cells and their protective role has been shown in vitro and in vivo. The principal function of these class of proteins is operate as molecular chaperones contributing to cellular homeostasis. As molecular chaperones HSPs promote the correct folding of nascent proteins and misfolded proteins, prevent the protein aggregation and assist protein transport across membrane (Gehrmann et al., 2008). Recently an additional roles have been associated to HSPs as danger signals and as potent activators of immune system. It has been shown that these molecules are 6 capable of inducing the production of proinflammatory cytokines by the monocyte-macrophage system and the activation of antigen-presenting cells (APCs) such as dendritic cells. In particular, mammalian cytosolic HSP70 and HSP90 bind antigenic peptides generated in cells and these complexes (HSP-PC) are taken up by antigen presentation cells via α2-macroglobulin receptor (CD91)-mediated endocytosis, resulting in representation by the major histocompatibility complex (MHC) class I molecules (Srivastava, 2002). The capability of HSPs, particularly HSP70 and HSP90, to operate as molecular carrier for antigenic determinants suggests the possible application of these molecules for production of conjugated vaccines (Robert, 2003). Lately, Kumaraguru et al. demonstrated that plant HSP70 mediated activation of mammalian immune system (Kumaraguru et al., 2003). Therefore it‟s feasible to consider the use of plant HSP70 as adjuvant molecules for vaccines production or for other pharmaceutical applications . Plants represent a renewable source and a safe system for production of molecules that can be used for human or animal vaccines. Although plants could provide several pharmaceutical molecules the limitation for their massive use is mainly due to the very low content of active molecules in plant bio-mass and to difficult and expensive purification processes. Recombinant DNA technologies permit to overcome these limits. Many works reported the use of plant systems for the expression of therapeutic molecules, such as antibodies, hormones, growth factor, blood components, human and animal vaccines (Ma et al., 2006). Recombinant proteins can be produced in several plant tissues or can be targeted to subcellular compartments such as endoplasmic reticulum (ER), chloroplast and mitochondria, where proteins are more stable, or intercellular space (apoplast). Latest 7 approach can be utilized to facilitate the recovery of recombinant proteins from roots exudates into hydroponic medium. This system, named rhizosecretion, has been used firstly by Borjsiuk and collaborators in 1999 to produce three different recombinant proteins in plant root exudates (Borjsiuk et al., 1999). In our work we used Nicotiana tabacum to induce constitutive expression of Arabidopsis HSP70. In particular we have genetically engineer tobacco to direct the recombinant HSP70 in the secretory pathway providing the HSP70 gene with a ER signal peptide from the abundant ER protein calreticulin of N. plumbaginifolia (Cal-HSP70). The presence of this signal peptide in the N terminal site of HSP70 should direct the recombinant protein to apoplasts and subsequently to the rhizosecretion pathway and consequently secrete the protein in the sterile hydroponic medium. Moreover, HSP70 targeted with the calreticulin signal peptide has been labelled with six histidine residues (His-tag) at its C terminus (Cal-HSP70-His). The His-tag signal will allow the rapid detection of His-tag HSP70 in transgenic tobacco by immunolocalization analysis with anti-His specific antibody. The recombinant genes (Cal-HSP70 and Cal-HSP70-His), were inserted into binary plant expression vector “pJazz”, under transcriptional control of CaMV35S constitutive promoter. The expression of HSP70 in transgenic tobacco was evaluated by Quantitative Real Time PCR analysis. The results showed that HSP70 gene is correctly transcribed both in leaves and in roots of transgenic plants, although at different levels. A further goal of our work was to purify the recombinant HSP70 from genetically engineered a strain of the yeast Saccharomyces cerevisiae. In this case a specific recognition sequence for enterokinase cleavage was added in 5‟of the start codon 8 (first ATG) to allow the excision of extra sequences after purification of HSP70 protein from yeast cells. S. cerevisiae combines the advantages of unicellular organism, such as ease of genetic manipulation and growth, with the capability of proteins processing that are typical for eukaryotic organism (protein folding, assembly and posttranslational modifications). As plants, yeast represent a safe system for the production of recombinant proteins, since there are no health risks arising from the presence of toxins or potential human pathogens. In fact this organism is recognized by the US Food and Drug Administration as an organism generally regarded as safe (Porro et al., 2005). Since 1995 there are many examples of recombinant proteins that were expressed in yeast, spanning from pharmaceutical products (insulin, interferon, erythropoietin, vaccines against hepatitis B) to industrial enzymes (used for treatments of food, feed, detergents and health care) (Porro et al., 2005). To obtain the expression of recombinant His-tag HSP70 in S. cerevisiae the protein coding gene was cloned under control of yeast inducible promoter GAL10 in the commercial vector named pESC-Trp (Stratagene). The production of His-tagged HSP70 was achieved using a 3 liters fermentator and yeast cells were disrupted by using the French Press. Proteins extracted from yeast cells were purified by immobilized metal affinity chromatography (IMAC). The purification level of His-tagged HSP70 by IMAC was assessed by SDS-olyacrylamide gel electrophoresis and Western Blot analyses.