Over-expression of recombinant human phospholipid scramblase 1 in E. coli and its purification from inclusion bodies.

Abstract:

:Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (*30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue "Stains-all" dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli.

journal_name

Biotechnol Lett

journal_title

Biotechnology letters

authors

Sahu SK,Gopala Krishna A,Gummadi SN

doi

10.1007/s10529-008-9797-z

subject

Has Abstract

pub_date

2008-12-01 00:00:00

pages

2131-7

issue

12

eissn

0141-5492

issn

1573-6776

journal_volume

30

pub_type

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