Disruption of the Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin.
Expression of recombinant human albumin (rHA) in Saccharomyces cerevisiae resulted in secretion of both mature albumin and a 45 kDa degradation product, comprising an N-terminal fragment of rHA with heterogeneous C-termini between residues 403 and 409 (Geisow et al., 1991). Site-directed mutagenesis of the human albumin gene (HA) to change Arg410 to Ala (R410A) caused a significant reduction in the amount of fragment produced. Mutation of the adjacent dibasic site Lys413 Lys414 had little effect in isolation, but in combination with the R410A mutation resulted in a further reduction in the amount of rHA fragment produced. This reduction could be duplicated with nature-identical rHA by disruption of the gene for an aspartyl protease (YAP3), alone or in conjunction with disruption of the KEX2 gene. Disruption of KEX2 alone did not result in any improvement in the degree of degradation of the rHA. Reduced degradation was also observed when an rHA-human growth hormone fusion protein was secreted from a yap3 strain, suggesting that such strains may have a general utility for heterologous protein secretion.
Kerry-Williams
S M
SM
Delta Biotechnology Ltd, Nottingham, U.K.
Gilbert
S C
SC
Evans
L R
LR
Ballance
D J
DJ
D000418:Albumins / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000737:chemistry; D043484:Proprotein Convertases; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism
D000418:Albumins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C499266:YPS1 protein, S cerevisiae