Expression of the mature and the pro-form of human sterol carrier protein 2 in Escherichia coli alters bacterial lipids.
Sterol carrier protein 2 (SCP2) is a protein that is believed to be involved in the intracellular transport of cholesterol and phospholipids. Expression in mammalian COS cells of a cDNA encoding SCP2 revealed that the mature protein is synthesized as a pro-form containing a 20-residue amino-terminal leader sequence. The function of this presequence is currently not known, and pro-SCP2 is generally not detected in tissues. In order to obtain large quantities of pro-SCP2 as well as the mature form of human SCP2, Escherichia coli expression plasmids were constructed. Both proteins were produced in high yield (10-30% of the total cell protein) and were found in the supernatant fraction after cell lysis. Recombinant human SCP2 and pro-SCP2 were purified to homogeneity by acid precipitation followed by ion-exchange chromatography. Both recombinant human SCP2 and pro-SCP2 had sterol exchange activity similar to that seen with SCP2 purified from rat liver. In addition, the lipid content of SCP2- and pro-SCP2-producing E. coli was analyzed. Acidic lipids were significantly increased in the transfected cells. Specifically, fatty acids were increased 2-3-fold, phosphatidylglycerol was increased 2-fold, and lipid A was increased 3-4-fold, while neutral lipids were decreased 2-3-fold as compared to control cells. This alteration of the lipid composition of E. coli expressing SCP2 or pro-SCP2 is consistent with the proposed role for SCP2 in intracellular lipid movement.
Matsuura
J E
JE
Du Pont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0400.
George
H J
HJ
Ramachandran
N
N
Alvarez
J G
JG
Strauss
J F
JF
Billheimer
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D013261:Sterols
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D010940:Plant Proteins; D011498:Protein Precursors; D013261:Sterols; C072087:sterol carrier proteins