Tareas

Protein Expression and Purification 21, 71– 80 (2001)
doi:10.1006/prep.2000.1335, available online at http://www.idealibrary.com on

Effect of Copy Number on the Expression Levels
of Hepatitis B Surface Antigen in the
Methylotrophic Yeast Pichia pastoris
Ana Vassileva, Dipti Arora Chugh, Sathyamangalam Swaminathan, and Navin Khanna
International Centre for Genetic Engineering andBiotechnology, P.O. Box 10504, Aruna Asaf Ali Marg,
New Delhi 110016, India

Received June 16, 2000, and in revised form August 17, 2000

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. pastorisexpression 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 HBsAgexpressing cassettescould 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 Mut s (methanol utilization slow) transformants that are known to have
an increased ability to generate HBsAg in particulate
form. A systematic investigation of theresultant
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 andsedimentation 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. © 2001 Academic Press
Key Words: hepatitis B surface antigen; Pichia pastoris.

tion and there are 350 million chronic carriers (1). As
there is no effective treatment for hepatitis B (2),
apreventive vaccine is the only method available for
its control. The surface antigen of hepatitis B virus
(HBsAg) 1 is the primary component of vaccines against
hepatitis B virus infection (3). To be immunogenic, the
HBsAg polypeptide must assemble into particles of
20 –22 nm diameter (4). HBsAg particle assembly
does not appear to occur in E. coli-based expression
systems (5–7). Commerciallyavailable recombinant
hepatitis B vaccine produced in Saccharomyces cerevisiae is quite expensive, especially due to the lower
expression levels of HBsAg (8). The high cost of the
recombinant vaccine is the single most important factor preventing its inclusion in the Expanded Program
on Immunization of developing countries for mass immunization. Higher expression levels may cut down
theproduction cost of this vaccine.
In recent years, the methylotrophic yeast Pichia pastoris has emerged as a powerful and inexpensive heterologous system for the production of high levels of
functionally active recombinant proteins (9 –11). As a
yeast, it is a unique system in that it combines the
advantages of both prokaryotic (high expression levels,
easy scale-up, inexpensive growth media) andeukaryotic (capacity to carry out most of the posttranslational
modifications characteristic of higher eukaryotes) expression systems (12). From an expression perspective,
the existence of well-established fermentation methods
that can generate very high cell densities using purely
defined media (13) and the strong, tightly regulated
methanol-inducible alcohol oxidase (AOX1) promoter
1...