Use of the cell wall protein Pir4 as a fusion partner for the expression of Bacillus sp. BP-7 xylanase A in Saccharomyces cerevisiae.
Xylanase A from Bacillus sp. BP7, an enzyme with potential applications in biotechnology, was used to test Pir4, a disulfide bound cell wall protein, as a fusion partner for the expression of recombinant proteins in standard or glycosylation-deficient mnn9 strains of Saccharomyces cerevisiae. Five different constructions were carried out, inserting in-frame the coding sequence of xynA gene in that of PIR4, with or without the loss of specific regions of PIR4. Targeting of the xylanase fusion protein to the cell wall was achieved in two of the five constructions, while secretion to the growth medium was the fate of the gene product of one of the constructions. In all three cases localization of the xylanase fusion proteins was confirmed both by Western blot and detection with Pir-specific antibodies and by xylanase activity determination. The cell wall-targeted fusion proteins could be extracted by reducing agents, showing that the inclusion of a recombinant protein of moderate size does not affect the way Pir4 is attached to the cell wall. Also, the construction that leads to the secretion of the fusion protein permitted us to identify a region of Pir4 responsible for cell wall retention. In summary, we have developed a Pir4-based system that allows selective targeting of an active recombinant enzyme to the cell wall or the growth medium. This system may be of general application for the expression of heterologous proteins in S. cerevisiae for surface display and secretion.
Andrés
Isabel
I
Unidad de Microbiología, Facultad de Farmacia, Univ. De Valencia, Avda. Vicente Andrés Estelles s/n. 46100-Burjassot (Valencia), Spain.
Gallardo
Oscar
O
Parascandola
Palma
P
Javier Pastor
F I
FI
Zueco
Jesús
J
D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases