Expression of human liver fatty acid-binding protein in Escherichia coli and comparative analysis of its binding characteristics with muscle fatty acid-binding protein.
Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia coli. The cDNA encoding human liver FABP was under the control of T7 RNA polymerase promoter in the expression vector pET-3b. Expression required overnight induction with isopropyl beta-D-thiogalactopyranoside in the presence of the bacterial RNA polymerase inhibitor, rifampicin. The protein could be purified by (NH4)2SO4 fractionation, anion-exchange and gel filtration chromatography, and was recognized by anti-(human L-FABP) antiserum. The binding characteristics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and for various hydrophobic ligands, were determined by radiochemical analysis and also by fluorescence for L-FABP. The apparent binding affinity of the ligands was calculated by using displacement curves of oleic acid and dansylamino-undecanoic acid (DAUDA). L-FABP showed a preference for the binding of long-chain saturated and unsaturated fatty acids up to C24:1, whereas the M-FABP has a preference for unsaturated fatty acids, especially those with 18 C atoms. L-FABP also binds palmitoyl derivatives and many other hydrophobic ligands--however, generally with a lower affinity than fatty acids. M-FABP binds--besides with fatty acids--only with oestradiol and testosterone with high affinity. Fatty acids with fluorescent reporter groups are also more tightly bound by L-FABP. A direct assay and displacement study of oleic acid gave the same Kd value of DAUDA for L-FABP. Fluorescence enhancement and displacement studies indicate that the binding of fluorescent fatty acids is determined by both the fluorescent reporter group and the acyl carbon chain.
Maatman
R G
RG
Department of Biochemistry, University of Nijmegen, The Netherlands.
van Moerkerk
H T
HT
Nooren
I M
IM
van Zoelen
E J
EJ
Veerkamp
J H
JH
D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003619:Dansyl Compounds / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000737:chemistry*; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D009363:Neoplasm Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011453:Prostaglandins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D025521:Tumor Suppressor Proteins; D014764:Viral Proteins
D002352:Carrier Proteins; D018076:DNA, Complementary; D003619:Dansyl Compounds; C493319:FABP1 protein, human; C107608:FABP7 protein, human; C493320:Fabp1 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009829:Oleic Acids; D011453:Prostaglandins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D014764:Viral Proteins; D019301:Oleic Acid; C054400:11-(dansylamino)undecanoic acid; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases