Biotechnological production of allergenic pectate lyase identified in cypress pollens

Pollen allergy is a serious health problem that has negative effects on people's health and causes economic losses in the world and in our country. Although allergenic pollens mostly affect the upper respiratory tract and eyes, they can trigger more serious diseases such as asthma. Thus early diagnosis and effective treatment are of great importance in pollen allergy. In recent years, pollen allergy, like all allergic diseases, has increased dramatically due to environmental factors such as air pollution, global warming, and intense urbanization. There is an urgent need for specific and standardized new products to be used in the diagnosis and treatment of pollen allergies, such as allergic rhinitis and conjunctivitis. Recombinant allergens show promise in the diagnosis and treatment of pollen allergy. Tree pollens contain many allergen proteins. Pectate lyase, an enzyme that breaks down polysaccharide chains containing galacturonic acid, found in cypress (Cupressaceae) pollens, is one of the allergenic proteins. In this study, obtaining a product that will be used in further studies and applications was aimed by recombinant production of the pectate lyase (Cup s 1) found in local cypress (Cupressus sempervirens) pollen in Escherichia coli. DH5α strain was used for plasmid (pQE-2) production, and E. coli BL21 (DE3) strain was used for recombinant protein production. A cDNA library was created by PCR from total RNA isolated from cypress pollen, and histidine-labeled Cup s 1 gene was amplified and ligated with plasmid by RT-PCR using cDNA as a template. Transfer of pQE-2 plasmid and recombinant plasmid to E.coli was performed by heat shock method and transformant cells were grown following their selection by colony PCR. Transformant cells were grown in a medium containing 1 mM IPTG ("isopropylthio-β-D-galactopyranoside") followed by precipitated and homogenized. The recombinant protein was purified by nickel affinity chromatography and analyzed by gel electrophoresis. The allergenicity of the pure recombinant protein was confirmed by Western blot analysis. As a result, biotechnological production of allergenic cypress pollen pectate lyase, which has the potential to be used in diagnosis or immunotherapy, has been successfully achieved.