Human phospholipid scramblase 1(hPLSCR1), when expressed in E. coli (BL-21 DE3), forms inclusion bodies that are functionally inactive. We studied the effects of various stress inducing agents and chaperones on soluble expression of hPLSCR1 in E. coli (BL-21 DE3). Addition of 3% (v/v) ethanol before induction and decreasing the post-induction temperature to 15°C increased the solubility of hPLSCR1 to ~10 and ~15% respectively. Presence of groES-groEL complex solubilized the hPLSCR1 to ~30% of the total hPLSCR1. Absence of groES-groEL did not improve the solubility of hPLSCR1 suggesting that groES and groEL are the essential chaperones for the correct folding of hPLSCR1 when over-expressed in E. coli. © Springer Science+Business Media B.V. 2009.

Sathyanarayana Naidu Gummadi

Department Of Biotechnology

Archita Rajasekharan

Chaperones

GROEL-GROES

HUMAN PHOSPHOLIPID SCRAMBLASE

Inclusion bodies

Membrane protein

Escherichia coli

Ethanol

Proteins

solubility

Ternary systems

Phospholipids

alcohol

CHAPERONIN

Phospholipid

PHOSPHOLIPID TRANSFER PROTEIN

PLSCR1 PROTEIN, HUMAN

recombinant protein

article

chemistry

drug effect

genetics

Human

metabolism

physiological stress

plasmid

Protein folding

protein quaternary structure

Temperature

CHAPERONIN 10

CHAPERONIN 60

Humans

PHOSPHOLIPID TRANSFER PROTEINS

Plasmids

Protein Structure, Quaternary

Recombinant Proteins

Stress, Physiological

IIT Madras is a public technical and research university located in Chennai, Tamil Nadu. Founded in 1959, it is recognised as an Institute of National Importance.

IIT Madras has been ranked as the top engineering institute in India for four years in a row by the National Institutional Ranking Framework of the MHRD

It currently offers undergraduate, postgraduate and research degrees across 16 disciplines in Engineering, Sciences, Humanities and Management. About 596 faculty belonging to science and engineering departments and centres of the Institute are engaged in teaching, research and industrial consultancy.

IIT Madras

Biotechnology Letters

Human phospholipid scramblase 1 (hPLSCR1) is an ATP independent, Ca2+ dependent transmembrane protein mediating bidirectional translocation of phospholipids across the lipid bilayer but the mechanism of scrambling is unknown. Determination of the hPLSCR1 structure would help understand the mechanism and its multi-functional property. Recombinant hPLSCR1 forms inclusion bodies (IBs), when over-expressed in E. coli BL21 (DE3) and recovery of active hPLSCR1 from IBs, were time-consuming and resulted in low protein yield. This study aims to optimize and enhance the expression and purification of active recombinant hPLSCR1 by various strategies. Additives including stabilizers and detergents were added during cell lysis to improve the recovery of soluble hPLSCR1. Five E. coli strains, BL21 (DE3), C43 (DE3), Rosetta, BL21-CodonPlus-RP, and BL21 (DE3) pLysS were screened for maximum yield of soluble protein but localized in IBs. To recover hPLSCR1 from IBs, different additives were added of which, 0.3% N-lauroyl sarcosine (NLS) recovered ∼50% of bioactive hPLSCR1 from IBs. E. coli C43 (DE3) gave higher yields of purified protein (7.76 mg/g cell) followed by E. coli BL21 (DE3) pLysS (5.87 mg/g cell). This report describes a rapid and efficient method for solubilizing membrane proteins from inclusion bodies with a higher recovery. © 2018 Elsevier Inc.

Analytical Biochemistry

Rapid method for an enhanced recovery of biologically active human phospholipid scramblase1 from inclusion bodies

Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca2+) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni2+-NTA) affinity chromatography and was&gt;98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ̃ 18% of 7-nitrobenz-2- oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca2+ ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an a-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques. © Society for Industrial Microbiology and Biotechnology 2012.

Journal of Industrial Microbiology and Biotechnology

Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine

Human phospholipid scramblase 1 (hPLSCR1) is a Ca2+-dependent protein known to scramble phospholipids in the plasma membrane resulting in loss of membrane asymmetry. It has been reported that hPLSCR1 exhibits Ca2+-independent activity at low pH. However, the conformational changes induced at low pH leading to functional activation are not known. Our results showed that recombinant hPLSCR1 was functionally activated at low pH, which is similar to the behavior of natively extracted hPLSCR1. Tryptophan fluorescence measurements showed a decrease in Ca2+-binding affinity at low pH, although not at pH 5.5. Far and near UV-CD revealed that low pH induced structural changes, with a significant increase in the β-sheet content of the protein. At the physiological level, decreased hPLSCR1 expression was observed after a period of exposure to low pH. The effect occurred at the promoter level. The expression levels of hPLSCR1 directly correlated with the sensitivity of HEK293 to apoptosis. Based on these results, we conclude that the mechanisms of Ca2+- and pH-induced functional activation of hPLSCR1 are different and that hPLSCR1 expression regulated by low pH could provide insights into the role of hPLSCR1 in cancer progression. © University of Wrocław, Poland 2015.

Fulltext

Cellular and Molecular Biology Letters

Biochemical evidence for Ca2+-independent functional activation of hPLSCR1 at low pH

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. © 2008 Springer Science+Business Media B.V.

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

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions. © 2007 Elsevier Inc. All rights reserved.

Archives of Biochemistry and Biophysics

Phospholipid scramblases: An overview

Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Hipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent ∼1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.

Biochemistry

Chemical modification identifies two populations of glycerophospholipid flippase in rat liver ER

Protein Expression and Purification

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

GroES and GroEL are essential chaperones for refolding of recombinant human phospholipid scramblase 1 in E. coli

Journal	Biotechnology Letters
ISSN	01415492
Open Access	No