Purification of recombinant human secretory phospholipase A2 (Group II) produced in long-term immobilized cell culture

Levin, Wayne; Daniel, R.; Stoner, Cheryl R.; Stoller, Timothy J.; Wardwell-Swanson, Judith; Angelillo, Yale M.; Familletti, Philip C.; Crowl, Robert M.

doi:10.1016/1046-5928(92)90052-x

Cited by 11 publications

(6 citation statements)

References 26 publications

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“…The expression vector encoding the human Group I1 PLA, was transfected into CHO cells and a stable cell clone secreting high levels of PLA, (ca 1 mg/L) was selected for further study. This level of rh-PLA, expression excedes those previously reported (Wery et al, 1991;Levin et al, 1992) and may relate to expression vector construction or cell culture conditions. The CHO cell clone secreting rh-PLA, was scaled up from spinner flasks to a 40 L reactor then to a 180 L reactor.…”

Section: Expression Of Rh-pla In Cultured Cho Cellssupporting

confidence: 73%

Recombinant human secretory phospholipase A₂: Purification and characterization of the enzyme for active site studies

Stadel

Jones

Livi

et al. 1992

J of Molecular Recognition

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A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.

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Section: Expression Of Rh-pla In Cultured Cho Cellssupporting

confidence: 73%

Recombinant human secretory phospholipase A₂: Purification and characterization of the enzyme for active site studies

Stadel

Jones

Livi

et al. 1992

J of Molecular Recognition

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“…644 was purified and characterized in detail. [648][649][650][651][652][653][654] The Ca 2+ -dependent enzyme has a molecular mass of 13.7 kDa, a pH-optimum at 9.0, and a characteristic substrate specificity (PE > PS > PC). Human synovial fluid contains at least two enzymatically distinct PLA 2 activities.…”

Section: Pla 2 In Joint Disordersmentioning

confidence: 99%

Phospholipase A₂: Its Usefulness in Laboratory Diagnostics

Kaiser

1999

Critical Reviews in Clinical Laboratory Sciences

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Phospholipase A2 (PLA2) is an enzyme that catalyzes the hydrolysis of membrane phospholipids. This article reviews the source and structure of PLA2, the involvement of the enzyme in various biological and pathological phenomena, and the usefulness of PLA2 assays in laboratory diagnostics. Of particular importance is the role of PLA2 in the cellular production of mediators of inflammatory response to various stimuli. Assays for PLA2 activity and mass concentration are discussed, and the results of enzyme determinations in plasma from patients with different pathological conditions are presented. The determination of activity and mass concentration in plasma is particularly useful in the diagnosis and prognosis of pancreatitis, multiple organ failure, septic shock, and rheumatoid arthritis. A very important result is the demonstration that PLA2 is an acute phase protein, like CRP. Indeed, there is a close correlation between PLA2 mass concentration and CRP levels in several pathological conditions. Although the determination of C-reactive protein is much easier to perform and is routinely carried out in most clinical laboratories, the assessment of PLA2 activity or mass concentration has to be considered as a reliable approach to obtain a deeper understanding of some pathological conditions and may offer additional information concerning the prognosis of several disorders.

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“…The sodium ion concentration was 250 mM Na + and the reaction time was 15 min. The purified human r-sPLA 2 used was prepared by W. Levin and R. M. Crowl (Hoffmann La-Roche Inc., Nutley, N J) [7].…”

Section: C42mentioning

confidence: 99%

Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2

1993

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Human cytosolic phospholipase A2 (cPLA2, 85 kDa) appears to be pharmacologically distinct from human secretory phospholipase A2 (sPLA2, 14 kDa). Marine natural products and PLA2 substrate and product analogs were potent inhibitors of human recombinant sPLA2 (r-sPLA2), whereas these compounds stimulated, weakly inhibited, or had no effect on cPLA2 activity from the human monocytic cell line U937. In contrast, within a series of seven reported calmodulin (CaM) antagonists tested, significant correlations among the rank order of potencies of these compounds as inhibitors of cPLA2, r-sPLA2, and a CaM-dependent phosphodiesterase were observed. The correlated inhibitory effects of the hydrophobic CaM antagonists on cPLA2 and sPLA2 may reflect a common feature (possibly a hydrophobic domain) shared by these two types of enzymes.

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Purification of recombinant human secretory phospholipase A2 (Group II) produced in long-term immobilized cell culture

Cited by 11 publications

References 26 publications

Recombinant human secretory phospholipase A₂: Purification and characterization of the enzyme for active site studies

Recombinant human secretory phospholipase A₂: Purification and characterization of the enzyme for active site studies

Phospholipase A₂: Its Usefulness in Laboratory Diagnostics

Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2

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Purification of recombinant human secretory phospholipase A2 (Group II) produced in long-term immobilized cell culture

Cited by 11 publications

References 26 publications

Recombinant human secretory phospholipase A2: Purification and characterization of the enzyme for active site studies

Recombinant human secretory phospholipase A2: Purification and characterization of the enzyme for active site studies

Phospholipase A2: Its Usefulness in Laboratory Diagnostics

Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2

Contact Info

Product

Resources

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Recombinant human secretory phospholipase A₂: Purification and characterization of the enzyme for active site studies

Recombinant human secretory phospholipase A₂: Purification and characterization of the enzyme for active site studies

Phospholipase A₂: Its Usefulness in Laboratory Diagnostics