High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A2.
Bee venom phospholipase A2 (BV-PLA2) is a hydrolytic enzyme that specifically cleaves the sn-2 acyl bond of phospholipids at the lipid/water interface. The same enzyme is also believed to be responsible for some systemic anaphylactic reactions in bee venom sensitized individuals. To study the structure/function relationships of this enzyme and to define the molecular determinants responsible for its allergenic potential, a synthetic gene encoding the mature form of BV-PLA2 was expressed in Escherichia coli. This enzyme was produced as a fusion protein with a 6xHis-tag on its amino-terminus yielding 40-50 mg of fusion protein per 1 of culture after metal ion affinity chromatography. A kallikrein protease recognition site was engineered between the 6xHis-tag and the amino-terminus of the enzyme allowing isolation of the protein with its correct N-terminus. Recombinant affinity purified BV-PLA2 was refolded, purified to homogeneity, and cleaved with kallikrein, resulting in a final yield of 8-9 mg of active enzyme per 1 of culture. The enzymatic and immunological properties of the recombinant BV-PLA2 are identical to enzyme isolated from bee venom indicating a native-like folding of the protein.
Dudler
T
T
Swiss Institute of Allergy and Asthma Research, Davos.
Chen
W Q
WQ
Wang
S
S
Schneider
T
T
Annand
R R
RR
Dempcy
R O
RO
Crameri
R
R
Gmachl
M
M
Suter
M
M
Gelb
M H
MH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001514:Bee Venoms / Q000201:enzymology / Q000235:genetics*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005813:Genes, Synthetic; D007610:Kallikreins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2; D010957:Plasmids; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D001514:Bee Venoms; D011993:Recombinant Fusion Proteins; D004247:DNA; D010741:Phospholipases A; D054467:Phospholipases A2; D007610:Kallikreins