International Journal of Life Sciences and Biotech
Recombinant Production of Hydrophobin DewA in Pichia pastoris and Determination of Its Functions
Authors: Alpgiray TURGUT, Ayşenur YAZICI, Mesut TAŞKIN, Serkan ÖRTÜCÜ
Abstract: Hydrophobins, found around the aerial hyphae and reproductive structures of fungi, are small amphipathic surfactant proteins. Due to these amphipathic properties, they can change the properties of the surfaces they are on. Thanks to these properties, hydrophilic surfaces can be obtained by imparting hydrophobic properties. However, the low production amounts in the studies conducted to date limit biotechnological studies. In the study, pPICZα-A vector was used for high protein expression, since its restriction maps, gene functions and intracellular replication mechanisms are known. P. pastoris was used for recombinant production due to its low amount of endogenous protein production, especially capable of performing posttranslational modifications. codon optimization was first performed after obtaining the gene sequence of the DewA protein from GenBank (Uniprot ID: P52750). Firstly, the secretion signal sequence region of this sequence and the stop codon of the gene were removed. Then, the cutting region of restriction enzymes (EcoRI and XbaI) that did not cut the resulting sequence was added to the sequence. After it was synthesized commercially, it was produced recombinantly by plasmid isolation, enzyme cutting reaction and ligation processes. It was then transferred to P.pastoris by electroporation method. Optimization of methanol induction was carried out at 0.5%, 1% and 1.5%. Protein isolation was performed by taking samples every 24 hours during the incubation. After protein isolation, the surface properties on teflon and glass surfaces were examined. 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. 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: HFBI Pichia pastoris silver staining, surface coating
dergipark.org.tr/tr/download/article-file/3388388
Recombinant Production of Hydrophobin DewA in Pichia pastoris and Determination of Its Functions
Authors: Alpgiray TURGUT, Ayşenur YAZICI, Mesut TAŞKIN, Serkan ÖRTÜCÜ
Abstract: Hydrophobins, found around the aerial hyphae and reproductive structures of fungi, are small amphipathic surfactant proteins. Due to these amphipathic properties, they can change the properties of the surfaces they are on. Thanks to these properties, hydrophilic surfaces can be obtained by imparting hydrophobic properties. However, the low production amounts in the studies conducted to date limit biotechnological studies. In the study, pPICZα-A vector was used for high protein expression, since its restriction maps, gene functions and intracellular replication mechanisms are known. P. pastoris was used for recombinant production due to its low amount of endogenous protein production, especially capable of performing posttranslational modifications. codon optimization was first performed after obtaining the gene sequence of the DewA protein from GenBank (Uniprot ID: P52750). Firstly, the secretion signal sequence region of this sequence and the stop codon of the gene were removed. Then, the cutting region of restriction enzymes (EcoRI and XbaI) that did not cut the resulting sequence was added to the sequence. After it was synthesized commercially, it was produced recombinantly by plasmid isolation, enzyme cutting reaction and ligation processes. It was then transferred to P.pastoris by electroporation method. Optimization of methanol induction was carried out at 0.5%, 1% and 1.5%. Protein isolation was performed by taking samples every 24 hours during the incubation. After protein isolation, the surface properties on teflon and glass surfaces were examined. 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. 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: HFBI Pichia pastoris silver staining, surface coating
dergipark.org.tr/tr/download/article-file/3388388