KlPMR1 inactivation and calcium addition enhance secretion of non-hyperglycosylated heterologous proteins in Kluyveromyces lactis.
The Kluyveromyces lactis KlPMR1 gene is the functional homologue of Saccharomyces cerevisiae PMR1 which encodes a Ca(2+)-ATPase localized in the Golgi apparatus. We studied the effects of KlPMR1 inactivation on the glycosylation and secretion of native and heterologous proteins in K. lactis. We used acid phosphatase, recombinant human serum albumin and alpha-glucoamylase from Arxula adeninivorans as reporter proteins. The Klpmr1Delta strain showed enhanced secretion of the heterologous proteins analyzed; the improved rHSA production did not result from enhanced transcription but rather involved increased translation and/or secretion efficiency. The growth rate of mutant cells resulted slower as compared to that of wild-type strain. The addition of 10mM calcium to the culture medium, however, not only completely relieved the growth defect of the mutant cells but also improved the rate of heterologous proteins production. Moreover, the addition of this ion in the culture medium of K. lactis did not suppress the glycosylation defects; this is an important difference with respect to S. cerevisiae where the glycosylation is partially restored by Ca(2+) addition. The Klpmr1Delta strain as a host offers thus an additional advantage for those cases requiring that the produced recombinant protein would not result hyperglycosylated.
Uccelletti
D
D
Department of Developmental and Cell Biology, University of Rome La Sapienza P.le A. Moro 5, 00185 Rome, Italy.
Farina
F
F
Mancini
P
P
Palleschi
C
C
D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D005285:Fermentation; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D007120:Immunochemistry; D007716:Kluyveromyces / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase; D000252:Calcium-Transporting ATPases; D005947:Glucose; D002118:Calcium