Biotechnological production of an allergenic lipid transfer protein

Allergy is a widespread disease worldwide, and its incidence gradually increases. The most important group of molecules leading allergic reactions by exciting the immune system diversely are proteins. Allergen proteins arise from various sources. One of the most well-known sources are aeroallergen pollens. Identification and biotechnological production of common allergens in pollens are not only important for basic science studies intending to enlighten molecular mechanism of allergy, but also take part in current research topics, since they lead to developments in allergy diagnosis and treatment. Non-specific lipid transfer proteins (LTPs) in plants are known to cause pollen and food allergies, that are common in especially Mediterranean countries. Allergic properties of nsLTPs in pollen vary according to plant species, geographic region and environmental conditions. No study has been conducted up to date on the nsLTP in Morus alba (white mulberry) pollen known to have high allergenicity. The main purpose of current study is the biotecnological production of nsLTP in M. alba pollen for further use. In the first step of the study, total RNA was isolated from M. alba pollens and a cDNA library was constructed using RT-PCR. The nsLTP gene in cDNA library was amplified with appropriate primers by PCR from this cDNA library and the accuracy of the gene sequence was confirmed by Sanger DNA sequencing method. The nsLTP gene was amplified by PCR with primers containing sequences of proper restriction enzyme breakpoints. PCR product was cloned into the pQE-2 vector carrying 6xHis tag, followed by E. coli BL21 (DE3) cells were transformed with this vector. Transformant E. coli cells were selected by the colony PCR method. In the second step of the study, working volume was increased and protein synthesis in transformant cells was induced with isopropylthio-β-D-galactopyranoside (IPTG) to produce high amount of recombinant nsLTP protein. Total proteins isolated after the cell collection and disruption were loaded onto the immobilized metal affinity chromatography (IMAC) system for the purification of his tagged nsLTP. Amount of recombinant protein eluted with imidazol was detected with bicinchoninic acid (BCA) method. Electrophoretic separations and analyses were carried out in order to trail nsLTP, and to test its IgE binding capacity, allergenicity, by Western blotting with the sera of patients allergic to M. alba. As a result, high amount of pure recombinant nsLTP having allergenic properties like natural protein was successfuly produced. Biotechnologial product obtained in this study can be used further basic and clinical studies for a better understanding the role of LTPs in allergy progression. Besides, it can be a part of specific diagnosis and treatment strategies and tools in the future.