The retention mechanism of cell wall proteins in Saccharomyces cerevisiae. Wall-bound Cwp2p is beta-1,6-glucosylated.

It has been proposed that the cell wall proteins of Saccharomyces cerevisiae are anchored by means of a beta-1,6-glucose-containing side chain. Recently, we have identified three cell wall mannoproteins. Two of these mannoproteins are recognized in their cell wall bound form by an antiserum raised against beta-1,6-glucan but the third, Cwp2p, is not. This could indicate the existence of alternative retention mechanisms for cell wall proteins. Western analysis of a fusion protein consisting of Cwp2p and the reporter enzyme alpha-galactosidase revealed that this protein is glycosyl phosphatidylinositol-anchored in the intracellular precursor form and is recognized by an anti beta-1,6-glucan antiserum in the cell wall bound form. The cell wall bound forms of fusion proteins consisting of the anchor regions of Sed1p or Flo1p and alpha-galactosidase were also recognized by an anti beta-1,6-glucan antiserum. This is consistent with the existence of a general anchoring mechanism of proteins to the cell wall by means of a beta-1,6-glucose-containing carbohydrate chain. Western analysis of a yeast strain producing c-myc epitope tagged Cwp2p revealed that this protein is only detectable if fatty acid chains are present on the protein, indicating that the lack of recognition of Cwp2p by an anti beta-1,6-glucan antiserum is caused by a blotting artefact of the mature protein.

Immobilizing proteins on the surface of yeast cells.

Yeast has a rigid cell wall comprising an outer layer of glycoproteins and an internal skeletal layer of glucan; heterologous proteins can be targeted to the glycoprotein layer and become covalently linked to the glucan skeleton. Yeast is a eukaryote that has 'generally regarded as safe' (GRAS) status, and is easy to cultivate, so it seems ideally suited for applications including the manufacture of recyclable, immobilized, biocatalysts, whole-cell vaccines, the presentation of peptide or antibody libraries, and the presentation of adhesion or metal-binding proteins.

Engineering surface charges in a subtilisin.

Introduction of multiple charged amino acid residues in the subtilisin Savinase by genetic engineering allowed us to modify the electrostatic properties of this enzyme in a systematic way. The effects of these charge changes were investigated theoretically with the calculated electrostatic potential at the enzyme surface and experimentally using ion exchange chromatography. Our results indicate that the effect of introducing charged residues at the enzyme surface depends on the local electrostatic potential. The effects are purely additive for residues that are not too closely packed at the enzyme surface. Although it is generally accepted that polarization effects are relatively small, our data show that substantial charge shifts arise when the dominating effect of the overall charge is taken away. These shifts are not well quantified using current methods to calculate the electrostatic potential at the enzyme surface. Our work focuses [correction of focusses] on methods that will provide a better description of this surface potential.

Expression of an alpha-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus.

For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).

Construction of a reshaped HMFG1 antibody and comparison of its fine specificity with that of the parent mouse antibody.

A human antibody with milk mucin specificity was obtained by transferring the complementarity determining regions (CDR) of the mouse antibody HMFG1 onto carefully selected human framework regions. The resulting reshaped human antibody, HuHMFG1, showed no difference in relative affinity for its antigen compared with the parent mouse HMFG1. The minimum epitope recognized by both the mouse and reshaped antibodies was demonstrated by epitope mapping to be identical, and consists of the tetramer PDTR. In a replacement net analysis, in which each of the amino acids was replaced in turn with the 19 other residues, it was determined that mouse HMFG1 and HuHMFG1 reacted with this series of synthetic peptides in an equivalent manner, indicating retention of identical fine specificity in the HuHMFG1 antibody. In contrast to other published reports, this was achieved without involvement of any framework residues in the binding site transfer. These data demonstrate that if well-matching human framework regions are employed grafting the CDR only can be sufficient to confer desired specificities to human antibodies and can, indeed, provide human analogues of mouse antibodies with virtually indistinguishable affinities and fine specificities relative to the mouse parent antibodies.

Organization, structure and expression of murine interferon alpha genes.

Using a human interferon-alpha probe we have isolated recombinant phages containing murine interferon-alpha (Mu IFN-alpha) genes from a genomic library. One of these phages contained two complete Mu IFN-alpha genes and part of a third gene. The insert of a second phage held two IFN genes. This indicates that the Mu IFN-alpha genes are clustered in the genome as is the case for the analogous human genes. The nucleotide sequences of these 5 genes were determined. They show that the genes are all different, albeit highly homologous. The deduced amino acid sequences show that four of the five genes contain a putative glycosylation site. Three genes were transiently expressed in COS cells and they gave rise to protein products showing antiviral properties. The expression of the five Mu IFN-alpha genes and the Mu IFN-beta gene was studied in virus-induced mouse L cells. The individual mRNAs were visualized in a nuclease S1 experiment, using a specific probe for each gene. In RNA preparations from induced cells mRNAs for each of the five alpha genes and the beta gene were present. However, substantial differences in the amounts of the individual mRNAs were observed.