Transformation of pPICZaA-E2 to Pichia pastoris X-33 and Mut⁺ phenotype analysis

Chikungunya is one of the current infectious, re-emerging diseases. Chikungunya virus (CHIKV) infection can cause some non-specific symptoms such as fever, arthralgia, and myalgia, similar to dengue and zika virus infection. The similarity of the symptoms frequently makes the diagnosis inaccurate. Therefore, a rapid and accurate diagnostic method such as an antibody-based Rapid Diagnostic Test (RDT) is needed to prevent a late or inaccurate diagnosis that can cause prolonged effects of infection. This study aims to obtain a recombinant clone colony of P. pastoris X-33 as an initial step for antibody-based RDT kit development. This study includes the transformation and the Mut⁺ phenotype confirmation of recombinant clones. The recombinant plasmid pPICZaA-E2 was transformed and integrated into P. pastoris X-33 genome using the electroporation method. After screening process through genome PCR, four positive recombinant clones colonies were analyzed in minimal dextrose and methanol media for Mut⁺ confirmation. Visualization of PCR colony shows two bands were formed in the size of 2.200 bp corresponding to the P. pastoris genome’s AOX1 gene and 1.848 bp corresponding to recombinant plasmid’s AOX1 gene that indicates the recombinant plasmid pPICZaA-E2 was integrated into the genome. All of the recombinant clones colonies grew well in minimal dextrose and minimal methanol media. Based on the results, it can be predicted that the Mut+ phenotype of P. pastoris recombinant clone was successfully obtained. However, gene sequencing must be done as a further step to confirm the presence of the recombinant DNA and the AOX1 gene in P. pastoris.