Buffered non-fermenter system for lab-scale production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae.
Expression of recombinant proteins using a secretion system can minimize co-purification of contaminating host proteins. Production of His-tagged recombinant proteins in the yeast alpha-factor secretion system has previously required a fermenter system to control the growth conditions such as pH of the yeast culture. We describe an inexpensive non-fermenter system for the production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae that uses a buffered low peptone YP glycerol medium, which does not interfere with immobilized metal affinity chromatography. Maspin, a tumor suppressor serpin, was expressed as a secreted N-terminal His/FLAG-tagged protein. Purification of the soluble active recombinant protein only requires centrifugation, concentration by ultrafiltration, and Ni2+ affinity chromatography. Purified protein yields of this system are 3-5 mg/L culture medium.
Ngamkitidechakul
Chatri
C
Medical College of Wisconsin, Milwaukee, WI, USA.
Twining
Sally S
SS
D002021:Buffers; D002498:Centrifugation; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media; D016147:Genes, Tumor Suppressor; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009532:Nickel; D009571:Nitrilotriacetic Acid; D009842:Oligopeptides; D010455:Peptides; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D015843:Serpins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014462:Ultrafiltration
D002021:Buffers; D003470:Culture Media; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C085344:SERPIN-B5; D015843:Serpins; C033223:polyhistidine; D006639:Histidine; D009532:Nickel; C085706:FLAG peptide; D009571:Nitrilotriacetic Acid