Functional characterization of human recombinant apolipoprotein AIV produced in Escherichia coli.
Apolipoprotein AIV (apoAIV), a protein which is known to activate the enzyme lecithin: cholesterol acyltransferase, to bind to apoAI/AII receptor sites and also to promote cholesterol efflux from adipose cells, may play an important role in reverse cholesterol transport. In this report, the high-level production of soluble recombinant mature human apoAIV (isoform 1) in Escherichia coli is described. The recombinant protein was purified by avoiding lipid extraction or denaturation. The apoAIV preparation was analysed by its reactivity with antibodies raised against human apoAIV, SDS-gel electrophoresis, isoelectric focusing and N-terminal sequencing. The purified recombinant protein retains an extra methionine at the N-terminus. Purified recombinant and natural apoAIV proteins were indistinguishable with regard to their denaturation properties, thermo-stability or their fluorescence emission properties in the presence of various quantities of a quenching agent. Complexes of ApoAIV with L-alpha-dimyristoyl-glycerophosphocholine (Myr2GroPCho), glycerophosphocholine (GroPCho), or L-alpha-1-palmitoyl-2-oleoylglycerophosphocholine (PamOleGroPCho) prepared from plasmatic and from recombinant apoAIV proteins have similar densities as revealed by analytical centrifugation. They also share the same cofactor properties for the lecithin:cholesterol acyltransferase reaction. Recombinant apoAIV complex with Myr2GroPCho was also able to bind to the same apoAI/AII receptor sites and to promote cholesterol efflux to an equal extent from adipose cells. It is concluded that the recombinant protein is functionally identical to the plasmatic apoAIV and may therefore be very useful in helping to elucidate the physiological role of apoAIV.
SERLIA, Institut Pasteur de Lille, France.
N
N
Duverger
A
A
Murry-Brelier
M
M
Latta
S
S
Reboul
G
G
Castro
J F
JF
Mayaux
J C
JC
Fruchart
J M
JM
Taylor
A
A
Steinmetz
P
P
Denèfle
D001053:Apolipoproteins / Q000096:biosynthesis; D001054:Apolipoproteins A / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D006358:Hot Temperature; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010957:Plasmids; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014461:Ultracentrifugation
D001053:Apolipoproteins; D001054:Apolipoproteins A; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; C022205:apolipoprotein A-IV; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase