Recombinant production of allergenic pectate lyase from cypress pollens

Abstract

Background/aim: Allergic rhinitis is a chronic disease becoming a major health problem, especially in metropolitan cities, mainly due to pollens. The Cupressus sempervirens (cypress) pollen is one of the most important causes of pollinosis, and it contains pectate lyase (Cup s 1) as a major allergen of approx. 40 kDa. The aim of this study was recombinant production of Cup s 1 in Escherichia coli for further use in the diagnosis and treatment of cypress pollen allergy.Materials and methods: Total RNA was isolated from C. sempervirens pollens (gathered in İstanbul), complementary DNA (cDNA) was synthesized, and then cDNA was used to amplify the Cup s 1 gene using PCR technique. The sequence of the Cup s 1 gene amplified by PCR was determined and verified by the BLAST. PCR product was then cloned into the pQE-2 vector. E. coli BL21 (DE3) cells were transformed with the vector for the recombinant protein synthesis. Recombinant protein was isolated from transformant cells, and purified by Ni2+ affinity chromatography. Subsequent to SDSPAGE, Coomassie staining as well as Western blotting were used for detecting recombinant protein band.Results: Recombinant Cup s 1 was successfully produced in E. coli and purified. Corresponding 40 kDa band was detected in the transformant cells, but not in the host cells following purification.Conclusion: Obtained recombinant allergen may be used in the diagnosis and/or treatment of cypress allergy after clinical trials in the future. This study is expected to provide a basis to similar attempts to improve the sensitivity and reliability of diagnosis of allergic diseases, and success of treatment in patients living in İstanbul.