Two-step cell disruption for the extraction of membrane-associated recombinant protein from Saccharomyces cerevisiae.
The use of rDNA technology to express heterologous proteins has been very successful during the last several years. Choice of an expression host is very important in order to retain the biological activity of recombinant proteins. Baker's yeast, Saccharomyces cerevisiae, is a eucaryotic GRAS organism suitable for the expression of biologically active proteins. Specifically, hepatitis B surface antigen (HBsAg) is expressed in baker's yeast. Because the yeast cells need to be disrupted for the recovery of bioactive intracellular proteins and because the protein HBsAg is hydrophobic and has a tendency to become associated with cell membranes, the use of detergent increases the recovery yield. In order to remove most of the contaminants from yeast, a two-step disruption/extraction scheme has been developed that facilitates downstream processing. Furthermore, it also has the advantage of minimizing proteolytic actions on the recombinant protein by removing most of the contaminants and proteases into the supernatant during the first disruption step, while keeping the desired protein in the pellet fraction. Final recovery is then achieved by the extraction process. Parameters affecting the disruption/extraction processes have been discussed.
Chi
W K
WK
Process Development Division, Development Center for Biotechnology, Taipei, Taiwan, Republic of China.
Ku
C H
CH
Chang
C C
CC
Tsai
J N
JN
D001709:Biotechnology; D002021:Buffers; D002458:Cell Fractionation / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; D005818:Genetic Engineering; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D002021:Buffers; D004274:DNA, Recombinant; D006514:Hepatitis B Surface Antigens; D008565:Membrane Proteins; D011994:Recombinant Proteins