Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.
Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.
Levin
W
W
Department of Protein Biochemistry, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Daniel
R F
RF
Stoner
C R
CR
Stoller
T J
TJ
Wardwell-Swanson
J A
JA
Angelillo
Y M
YM
Familletti
P C
PC
Crowl
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D046508:Culture Techniques / Q000379:methods; D004247:DNA / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification*; D054467:Phospholipases A2; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D004247:DNA; D010741:Phospholipases A; D054467:Phospholipases A2