Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae.
A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania, 19486, USA.
J C
JC
Cook
L D
LD
Schultz
J
J
Huang
H A
HA
George
W K
WK
Herber
C
C
Ip
J G
JG
Joyce
S S
SS
Mao
H Z
HZ
Markus
W J
WJ
Miller
M K
MK
Sardana
E D
ED
Lehman
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005822:Genetic Vectors; D036341:Intercellular Signaling Peptides and Proteins; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013987:Ticks / Q000235:genetics
D060829:Arthropod Proteins; D004274:DNA, Recombinant; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C063967:tick anticoagulant peptide; D000072235:Mating Factor