Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris

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 h eterologous gene expression cassette was created using an in vitro multimer ization approach. Using this strategy, constructs containing up to a maximu m of eight direct repeats of the HBsAg-expressing cassettes could be create d. These expression cassettes were targeted for integration into the genome of the host strain GS115 with simultaneous elimination of the resident AOX 1 gene. Deletion of the AOX1 gene was intended to create Mut(s) (methanol u tilization slow) transformants that are known to have an increased ability to generate HBsAg in particulate form. A systematic investigation of the re sultant clones demonstrated that the increase in copy number results in a p roportional elevation in the steady-state levels of the HBsAg-specific mRNA , which in turn is closely paralleled by a corresponding increase in the to tal levels of the HBsAg protein. Virtually all the recombinant protein in t he soluble fraction was present in the particulate form based on particle-s pecific ELISA and sedimentation behavior. Further, our studies also reveale d the continued physical and functional integrity of the HBsAg-expressing c assettes during the course of an extended induction phase spanning 6 days. (C) 2001 Academic Press.