Effect of copy number on the expression levels of Hepatitis B surface Antigen in the methylotrophic yeast Pichia pastoris

Abstract

High-level expression and efficient assembly of hepatitis B surface Antigen (HBsAg) has been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under control of the AOX1 promoter. To fully utilize the expression potential of the P. pastoris expression system, we investigated the influence of gene copy number on the expression of HBsAg in this yeast. A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. Using this strategy, constructs containing up to a maximum of eight direct repeats of the HBsAg-expressing cassettes could be created. These expression cassettes were targeted for integration into the genome of the host strain GS115 with simultaneous elimination of the resident AOX1 gene. Deletion of the AOX1 gene was intended to create Muts (methanol utilization slow) transformants that are known to have an increased ability to generate HBsAg in particulate form. A systematic investigation of the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the steady-state levels of the HBsAg-specific mRNA, which in turn is closely paralleled by a corresponding increase in the total levels of the HBsAg protein. Virtually all the recombinant protein in the soluble fraction was present in the particulate form based on particle-specific ELISA and sedimentation behavior. Further, our studies also revealed the continued physical and functional integrity of the HBsAg-expressing cassettes during the course of an extended induction phase spanning 6 days.