Genetic Stability Test of Pichia pastoris Carrying Integrated Pre-membrane Envelope Gene of DENV3 Indonesian Strain

Abstract

In recent years, the methylotrophic Pichia pastoris has become an increasingly famous host for recombinant protein expressions. As a eukaryote, this yeast offers several advantages, including simplicity of genetic manipulation, stable expression, and low-cost scalable fermentation techniques. Previous study has confirmed the insertion of Dengue Virus Serotype 3 (DENV3) pre-Membrane Envelope (pr-M/E) gene in the recombinant P. pastoris X33 generated in our laboratory. The study has also confirmed the strain's ability to express the protein.In this study, we are reporting the genetic stability of the recombinant strain, confirming the steady expression of the heterologous protein in subsequent generations. The genetic stability test was performed by PCR and DNA sequencing on the recombinant P. pastoris cultured in non-baffled shake flasks. Generation time was estimated based on the yeast growth curve and calculated using a previously published formula. According to the growth curve, the generation time of this correlates recombinant yeast is four hours. It differs from the wild type, which took 4.3 h to complete. PCR of target gene performed at generation 1, 18, 39, 56, 81, and 100 revealed two DNA bands which indicating the presence of full AOX1 gene (2.2 kb) and AOX1 promoter plus pr-M/E gene (2 kb). In addition, sequencing of the PCR products show only minor variation, which might have been genuine or a result of PCR or sequencing errors. However, because the amino acid sequences of generation 100 differed from neither the RefSeq nor the original plasmid, we predict that our recombinant P. pastoris stably contains the gene of interest.