Over-expression of recombinant human phospholipid scramblase 1 in E. coli and its purification from inclusion bodies.
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.
Sahu
Santosh Kumar
SK
Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600036, India.
Gopala Krishna
A
A
Gummadi
Sathyanarayana N
SN
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium; D002232:Carbocyanines / Q000378:metabolism; D002846:Chromatography, Affinity; D004227:Dithionite; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014508:Urea
D002232:Carbocyanines; D009942:Organometallic Compounds; C493422:PLSCR1 protein, human; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D049294:Phospholipid Transfer Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; C052267:1,2-(palmitoyl-NBD-aminocaproyl)phosphatidylethanolamine; D004227:Dithionite; C088321:nickel nitrilotriacetic acid; C012040:stains-all; C044745:1-palmitoyl-2-(6-(N-(7-nitrobenz)-2-oxa-1,3-diazol-4-yl)aminocaproyl)phosphatidylcholine; D014508:Urea; D009327:4-Chloro-7-nitrobenzofurazan; D009571:Nitrilotriacetic Acid; D002118:Calcium