Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life

Suzanne Barmada; Eric Kienle; Dennis R. Koop

doi:10.1006/bbrc.1995.1085

Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life

Suzanne Barmada, Eric Kienle, Dennis R. Koop

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

Abstract

Rabbit P450 2E1 was stably expressed in Chinese hamster ovary cells after cotransfection with pRC/CMV-2E1 and pFR400 which expresses murine dihydrofolate reductase with a single arginine to leucine substitution at position 22. This mutation permits amplification of expression with increasing methotrexate concentrations in CHO-K1 cells that are not dihydrofolate reductase deficient. After amplification with 1 μM methotrexate, a representative clone expressed about 15 pmol of P450 2E1/mg microsomal protein. Cells from a single 35-mm plate catalyzed the formation of 1.02 nmol 6-hydroxychlorzoxazone/10⁶ cells/h or about 127 pmol/mg total cell protein/min. The enzyme was rapidly labeled when pulsed with [³⁵S]-methionine. Initial pulse-chase experiments indicate that the expressed protein has a half-life of 4.8 h.

Original language	English (US)
Pages (from-to)	601-607
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	206
Issue number	2
DOIs	https://doi.org/10.1006/bbrc.1995.1085
State	Published - Jan 17 1995
Externally published	Yes

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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title = "Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life",

abstract = "Rabbit P450 2E1 was stably expressed in Chinese hamster ovary cells after cotransfection with pRC/CMV-2E1 and pFR400 which expresses murine dihydrofolate reductase with a single arginine to leucine substitution at position 22. This mutation permits amplification of expression with increasing methotrexate concentrations in CHO-K1 cells that are not dihydrofolate reductase deficient. After amplification with 1 μM methotrexate, a representative clone expressed about 15 pmol of P450 2E1/mg microsomal protein. Cells from a single 35-mm plate catalyzed the formation of 1.02 nmol 6-hydroxychlorzoxazone/106 cells/h or about 127 pmol/mg total cell protein/min. The enzyme was rapidly labeled when pulsed with [35S]-methionine. Initial pulse-chase experiments indicate that the expressed protein has a half-life of 4.8 h.",

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AU - Barmada, Suzanne

AU - Kienle, Eric

AU - Koop, Dennis R.

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AB - Rabbit P450 2E1 was stably expressed in Chinese hamster ovary cells after cotransfection with pRC/CMV-2E1 and pFR400 which expresses murine dihydrofolate reductase with a single arginine to leucine substitution at position 22. This mutation permits amplification of expression with increasing methotrexate concentrations in CHO-K1 cells that are not dihydrofolate reductase deficient. After amplification with 1 μM methotrexate, a representative clone expressed about 15 pmol of P450 2E1/mg microsomal protein. Cells from a single 35-mm plate catalyzed the formation of 1.02 nmol 6-hydroxychlorzoxazone/106 cells/h or about 127 pmol/mg total cell protein/min. The enzyme was rapidly labeled when pulsed with [35S]-methionine. Initial pulse-chase experiments indicate that the expressed protein has a half-life of 4.8 h.

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Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life

Abstract

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