High-level expression and purification of human leukotriene A4 hydrolase from insect cells infected with a baculovirus vector.
Leukotrienes constitute a group of bioactive compounds derived from arachidonic acid which play important roles in immediate hypersensitivity and inflammation. Leukotriene A4 hydrolase (LTA4H) is an epoxide hydrolase, catalyzing the hydration of LTA4 to LTB4, and also acts an aminopeptidase, with the ability to cleave amides of p-nitroaniline. The cDNA for LTA4H was cloned using oligonucleotide-directed amplification of the cDNA sequence by polymerase chain reaction and by oligonucleotide-based screening of a bacteriophage lambda gt11 cDNA library derived from human placental tissue. High levels of biologically active LTA4H were expressed in cultured Spodoptera frugiperda insect cells infected with a baculovirus expression vector containing the LTA4H cDNA. Expression levels were approximately 100 mg per liter of cell-free culture media. LTA4H was recovered from the medium and purified to > 95% purity by ion-exchange and gel-filtration chromatography, with an overall yield of 76%. LTA4H produced by insect cells exhibits both hydrolase and aminopeptidase activities and has kinetic properties similar to those reported for enzyme isolated from human lung. Two major isoforms, with pI's of 5.3 and 5.1, were isolated by preparative chromatofocusing chromatography. NH2-terminal sequence analysis revealed that the two different by an NH2-terminal blocking group. Electrospray ionization mass spectrometry indicates that the two isoforms differ by a molecular mass of 42, indicating that the blocking group is an acetyl group.
Gierse
J K
JK
Monsanto Corporate Research, Chesterfield, Missouri 63198.
Luckow
V A
VA
Askonas
L J
LJ
Duffin
K L
KL
Aykent
S
S
Bild
G S
GS
Rodi
C P
CP
Sullivan
P M
PM
Bourner
M J
MJ
Kimack
N M
NM
D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000235:genetics; D004851:Epoxide Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase