Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences

Abstract

We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively.

Expression of Human Gelatinase B in Pichia pastoris

“…As the copy numbers of the integrated GelB clones are similar, the enhanced secretion from GS115/pPIC9K-dGelB is most likely the result of substitution of the Mat ␣ signal peptide. This result is consistent with others suggesting that the type of the signal sequence affects the level of secretion of a foreign protein in P. pastoris (20).…”

Variation in N-Linked Oligosaccharide Structures on Heterologous Proteins Secreted by the Methylotrophic YeastPichia pastoris

“…Dextranase (11), ␣-amylase (12), Boophilus microplus gut (Bm86) antigen (13), and aspartic protease and invertase (unpublished results) glycoproteins were obtained from the Center for Genetic Engineering and Biotechnology (CIGB, Cuba), BioIndustry Division. Invitrogen Co. (San Diego, CA) supplied the pure bovine enterokinase catalytic subunit (EK L ) (14).…”

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

“…Extensive development of P. pastoris biotechnology over the past 10 years now permits the construction of efficient host-vector systems for heterologous protein expression. A number of proteins that are insoluble in E. coli have been expressed in an active and soluble form in P. pastoris (23), as have various eukaryotic proteins (7,9,13,26,29,30,32,39). P. pastoris possesses the complex eukaryotic secretion machinery and can secrete proteins containing a high number of disulfide bridges.…”