Expression of recombinant psoriasis-associated fatty acid binding protein in Escherichia coli: gel electrophoretic characterization, analysis of binding properties and comparison with human serum albumin.
The psoriasis-associated fatty acid binding protein (PA-FABP, also known as FABP5) is a novel keratinocyte protein that is highly up-regulated in psoriatic plaques (P. Madsen, H. H. Rasmussen, H. Leffers, B. Honoré and J. E. Celis, J. Invest. Dermatol. 1992, 99, 299-305). Here we have expressed PA-FABP in Escherichia coli as a fusion protein containing an NH2-terminal hexa-His tag followed by a factor Xa cleavage site. The recombinant protein was expressed at a level of about 30% of the soluble proteins and was purified to homogeneity using a simple two-step protocol consisting of affinity chromatography on Ni2+-nitrilotriacetic acid agarose followed by gel filtration. The recombinant protein was then digested with factor Xa and characterized by two-dimensional gel electrophoresis. The ability of PA-FABP to bind saturated fatty acids ranging from 6 to 16 carbons was determined directly by dialysis and compared to human serum albumin (HSA). The results showed that PA-FABP binds multiple molecules of the fatty acids hexanoate (C6:0), octanoate (C8:0), decanoate (C10:0) and laurate (C12:0), all with a K1 of about 10(4) M(-l), and myristate (C14:0) with a K1 of 4.4 X 10(5) M(-l). Palmitate (C16:0) also bound strongly with multiple molecules. Due to the very low solubility of palmitate its affinity to PA-FABP was measured relatively to HSA and found to be 8.1 times lower. At ligand/protein ratios below 1, all fatty acids bound to PA-FABP with about one to three orders of magnitude lower affinity than to HSA. The difference in the fatty acid binding properties of the two proteins may reflect differences in their three-dimensional structures, which in the case of PA-FABP consists mainly of beta-sheets while HSA contains predominantly alpha-helices.
Vorum
H
H
Department of Medical Biochemistry and Danish Centre for Human Genome Research, University of Aarhus.
Madsen
P
P
Svendsen
I
I
Cells
J E
JE
Honoré
B
B
D000418:Albumins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003651:Decanoates / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D006801:Humans; D007848:Laurates / Q000378:metabolism; D008969:Molecular Sequence Data; D018996:Myelin P2 Protein / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009226:Myristates / Q000378:metabolism; D009363:Neoplasm Proteins; D010168:Palmitates / Q000378:metabolism; D011487:Protein Conformation; D011565:Psoriasis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D025521:Tumor Suppressor Proteins
D000418:Albumins; D002352:Carrier Proteins; D003651:Decanoates; C493412:FABP5 protein, human; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D007848:Laurates; D018996:Myelin P2 Protein; D009226:Myristates; D009363:Neoplasm Proteins; D010168:Palmitates; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins