High-yield secretion of recombinant gelatins by Pichia pastoris.
Recombinant non-hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae alpha-mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3.0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono-arginylic sites, by a putative Kex2-like protease, could be successfully abolished by site-directed mutagenesis of these sites. Production levels as high as 14.8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris.
Werten
M W
MW
Agrotechnological Research Institute (ATO-DLO), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands. m.w.t.werten@ato.dlo.nl
van den Bosch
T J
TJ
Wind
R D
RD
Mooibroek
H
H
de Wolf
F A
FA
D000595:Amino Acid Sequence; D003094:Collagen / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005780:Gelatin / Q000032:analysis / Q000378:metabolism*; D005822:Genetic Vectors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry; D043484:Proprotein Convertases; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis; D013381:Subtilisins / Q000737:chemistry; D014170:Transformation, Genetic
D017931:DNA Primers; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005780:Gelatin; D003094:Collagen; D004247:DNA; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae