Pichia pastoris'te Hidrofobin DewA'nın Rekombinant Üretimi ve Fonksiyonlarının Belirlenmesi

Abstract

Hidrofobinler, değişen yüzey özellikleri nedeniyle birçok biyoteknolojik uygulamada büyük potansiyele sahiptir. Son yıllarda hidrofobinlerin biyoteknolojik uygulamalarında önemli bir artış olmasına rağmen verim sorunları nedeniyle hala endüstriyel üretim sağlanamamıştır. Bu nedenle hidrofobinlerin rekombinant üretimi konusunda daha fazla çalışmaya ihtiyaç vardır. Bu çalışmada, literatürde yüksek temas açısına sahip olduğu belirlenen Aspergillus nidulans'tan sınıf I hidrofobin DewA'nın rekombinant üretimi amaçlandı. Sonuç olarak DewA proteini, AOX1 promoteri altında P. pastoris X-33 suşu kullanılarak pPICZa-A vektörüne aktarılarak rekombinant şekilde üretildi. DewA ekspresyonu için optimal kültür koşulu, %1 metanol konsantrasyonunda 96 saatte 77 mg/L olarak elde edildi. Rekombinant DewA'nın teflon ve cam yüzeylerdeki yüzey özelliklerini değiştirdiği kanıtlanmıştır. Daha sonra bu yüzeylere sıcak SDS ve UV uygulanarak proteinin yüzey stabilitesi değerlendirildi. Yüzey kaplamalı DewA, hem cam hem de teflon yüzeylerde sıcak SDS uygulamasına dayanıklıydı; UV uygulamasında proteinin cam yüzeylerde UV ışınlarına maruz kalmasıyla bozunurken, teflon yüzeylerde ise yapısını koruduğu anlaşıldı. Çalışmada A.nidulans'ın DewA proteini, pPICZa-A vektörüne klonlandı ve ilk kez P.pastoris X-33 suşunda rekombinant olarak üretildi.

Keywords

• Aspergillus nidulans
• DewA
• Pichia pastoris
• Hidrofobin
• Rekombinant üretim

Recombinant Production of Hydrophobin DewA in Pichia pastoris and Determination of Its Functions

Abstract

Background Hydrophobins have great potential in many biotechnological applications due to changing surface characteristics. In recent years, although there has been a significant increase in the biotechnological applications of hydrophobins, industrial production has still not been achieved due to yield problems. Therefore, more studies are needed on the recombinant production of hydrophobins. In this work, the recombinant production of class I hydrophobin DewA from Aspergillus nidulans, which is determined to have high contact angle in the literature, was aimed. As a result, DewA protein was recombinantly produced using P. pastoris X-33 strain under AOX1 promoter by transferring into pPICZα-A vector. Results The optimal culture condition for DewA expression was obtained at 1% methanol concentration as 77 mg/L in 96 hour. Recombinant DewA has been proven to change the surface characteristics on the teflon and glass surfaces. Then, the surface stability of the protein was evaluated by applying hot SDS and UV to these surfaces. The surface-coated DewA was resistant to hot SDS application on both glass and teflon surfaces; in the UV application, it was understood that while the protein was degraded by UV exposure on glass surfaces, it preserved its structure on teflon surfaces. Conclusions In the study, the DewA protein of A.nidulans was cloned into the pPICZα-A vector and recombinantly produced in the P.pastoris X-33 strain for the first time.

Keywords

• Aspergillus nidulans
• DewA
• Pichia pastoris
• Hydrophobin
• Recombinant production

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