A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain.
A new high-yield yeast expression/secretion system has been adapted for the plant thiol endoprotease papain. The propapain gene, obtained from Carica papaya fruit, is expressed in the yeast Saccharomyces cerevisiae. The gene was cloned into a FLAG epitope-tagging expression vector downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence. Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2). Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture supernatant where it accumulates to approximately 1.7 mg/l. The proregion contains three consensus N-linked glycosylation sites, whereas there are only two such sites in previously reported cDNA sequences. Removal of this third N-linked glycosylation site results in a drastic reduction in the level of protease activity present in the culture supernatant. Two different types of affinity chromatography were used to purify either propapain or papain. The propapain precursor is autoproteolytically activated to mature papain (M(r) = 24 kDa) using conditions reported previously. The kinetic parameters obtained agree well with the literature values. The yields of active papain are 10-fold higher than those previously reported for propapain in other yeast or bacterial expression systems. This, together with the ease with which mutant proteins can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.
Ramjee
M K
MK
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3206, USA.
Petithory
J R
JR
McElver
J
J
Weber
S C
SC
Kirsch
J F
JF
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D004792:Enzyme Precursors; D010940:Plant Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; C071653:propapain; D010206:Papain