Use of the cell wall protein Pir4 as a fusion partner for the expression of Bacillus sp. BP‐7 xylanase A in Saccharomyces cerevisiae

Abstract

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.

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…In the case of Cwp1, the PIR repeat may be used as an additional wall anchorage point because the protein is attached to the wall by both an alkali-labile and a GPI-dependent linkage (Kapteyn et al 2001). Like GPI-CWP, PIR proteins can be used as carriers to direct surface expression of heterologous proteins fused to them (Andrés et al 2005;Shimma et al 2006).…”

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

“…For this, the DNA sequence coding VP8* was fused to the genes coding two cell wall proteins of S. cerevisiae, Icwp (Ssr1p) and Pir4. Icwp had been previously used by our group as a partner for the targeting to the cell wall of the Staphylococcal protein A (Andrés et al, 2003), whilst Pir4 has been recently being proposed as partner to promote the cell wall targeting or the secretion of recombinant enzymes (Andrés et al, 2005).…”

“…Constructions V2-V4 involved the fusion of VP8* with Pir4 a cell wall protein that can be extracted from the cell wall by reducing agents (Moukadiri et al, 1999). Also in this case, our group has shown the feasibility of using Pir4 to achieve the targeting to the cell wall or the secretion to the growth medium of recombinant proteins (Andrés et al, 2005). Constructions V2 and V3 consisted in the insertion of the VP8* sequence without affecting the Pir4 domain responsible for the attachment through disulphide bridges, whilst V4 involved replacing the region containing this domain by VP8*.…”

High cell density culture with S. cerevisiae CEN.PK113-5D for IL-1β production: optimization, modeling, and physiological aspects

“…In the present work, to discriminate the effects given by a heterologous metabolic burden over those related to the cultural conditions, we have compared the growth parameters of CEN.PK113-5D expressing an heterologous product against those of the non-transformed strain. The recombinant strain carries, on the episomal pIA1 derivative [7], the coding sequence for human interleukin-1b (IL-1b) gene fused with the regulatory sequences of PIR4 gene [8] that promotes IL-1b secretion to the growth medium [9].…”