Recombinant Expression And Purification Of House Dust Mite Allergens Der P 1 And Der P 23

Dust mite allergens are one of the most common allergens that cause human allergies.The house dust mite Der p 1,Der p 2 and Der p 23 proteins are important allergens causing allergies,and IgE antibodies that bind specifically to these three allergens can be detected in the serum of more than 70%allergic individuals.Dust mite allergens are mainly used for allergy detection and desensitization of allergy patients,so high purity of dust mite allergens is required.Traditional preparation of dust mite allergens is mainly obtained by chemical extraction of mites,but the obtained allergens are often complex in composition and low in purity.The recombinant allergens have the advantages of simple production process,high purity and abundant availability,and gradually become the preferred methods for natural allergens.In China,allergy testing starts late and mainly relies on imported allergens at present.Therefore,it is particularly important to develop the production technology of allergens and realize self-sufficiency.In this study,house dust mite allergens Der p 1 and Der p 23 were selected as the research targets.Pichia pastoris was used to express the allergens with the purpose of develop high-yield and high-purity production process of the recombinant allergens.Firstly,we selected the commercial expression host GS115 and the patented chassis strain GS_ΔkE1 constructed by our laboratory as the host,respectively,and constructed and screened the recombinant expression engineering strains of Der p 1 and Der p 23.The results showed that the highest expression levels of Der p 1 and Der p 23 reached 100 mg/L and 82 mg/L,respectively.Subsequently,with increase of the target gene copy number and co-expression of chaperones,the expression capacity of target proteins was further improved,and the maximum expression levels of Der p 1 and Der p 23 were increased to 140 mg/L and 225.4 mg/L.On this basis,the high-yielding strains were tested in a 3 L reactor,and the expression levels of the two recombinant allergens reached 1 g/L and 5 g/L,respectively.Secondly,the purification process of two recombinant allergens Der p 1 and Der p 23 was explored,and a simple and efficient purification process was established.Der p 1 was purified by cation exchange chromatography.The target protein was captured by SP Sepharose FF and eluted effectively with 500 mM sodium chloride,but there was still a small amount of impurity protein.Then target protein was captured by affinity medium Ni-TED and eluted by 40 mM imidazole.After two-step purification,the recovery of target product reached 60%,and high purity protein was obtained.Then,Der p 23 was purified by affinity chromatography,and the target protein was effectively bound to the affinity medium Ni-TED and eluted under 40 mM imidazole.By one step affinity chromatography,the target protein was effectively purified with a yield of 80.7%.Finally,we tested the activity of two recombinant allergens respectively and compared them with imported products.The activity of purified Der p 23 samples was almost identical with that of imported samples and can be used as a substitute for imported samples.However,the activity of Der p 1 was lower than that of the imported sample.The molecular weight analysis showed that the actual molecular weight of Der p 1 was 29 kDa,slightly larger than the theoretical molecular weight of 25 kDa.It was speculated that the incomplete treatment of the leading peptide led to changes of the protein conformation and affected activities of the protein.In conclusion,this project successfully developed recombinant expression strains,culture and purification process for production of recombinant dust mite allergens,and high yield and purity of products were obtained.The produced Der p 23 showed almost the same activity as the commercial product.It provides references for development of other recombinant dust mite allergens in P.pastoris.