Influence
of Signal Peptide Sequences on the Expression
of Heterogeneous Proteins in Pichia pastoris
XIONG Ai-Sheng, PENG Ri-He, LI
Xian, FAN Hui-Qin, YAO Quan-Hong*
GUO Mei-Jin1, ZHANG Si-Liang1
( Agro-Biotechnology Research Center of Shanghai Academy of
Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and
Breeding, Shanghai 201106, China;
1 Bio-engineering Department of East China University of Science and
Technology, Shanghai 200237, China )
Abstract Pichia pastoris has been developed to be a
very efficient expression host for the heterogeneous proteins since its alcohol
promoter was isolated and cloned, and its transformation technique was
established. For further improving the secretion expression of heterogeneous
proteins, in this research, the signal sequences were studied. At first, the Saccharomyces
cerevisiae mating factor &agr; prepro-leader sequence was synthesized
using successive PCR and designated as MF4I. Then, ten different signal
sequences were constructed by adding the N-terminal residues of Pichia
pastoris Aox1 protein to the N-terminal of the MF4I. These ten signal
sequences were used for directing phytase gene secretion in Pichia pastoris,
the secretion of phytase were increased in Pichia pastoris strains
containing new signal sequence. Among these strains, the phytase secretion was
highest in strain contain signal sequence added with A, I, P three Aox1
N-terminal residues; the phytase secretion of Pichia pastoris was 90
mg/L in flake. The secretion was six-fold of that with original Saccharomyces
cerevisiae mating factor &agr; prepro-leader sequence. In addition,
insert of ten residues E E A E A E A E P K can further increase the phytase
secretion by 35%, the secretion reach 120 mg/L.
Key
words
Pichia pastoris;
synthetic signal sequence; expression vector; high expression
Corresponding
author: Tel,
86-21-62208660-3207; Fax, 86-21-62209988; e-mail, [email protected]