One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli.
Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14-15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H-FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30-40 mg/l culture. Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins. In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP.
Schaap
F G
FG
Department of Physiology, University of Limburg, Maastricht, Netherlands.
Specht
B
B
van der Vusse
G J
GJ
Börchers
T
T
Glatz
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008297:Male; D008969:Molecular Sequence Data; D018996:Myelin P2 Protein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009206:Myocardium / Q000737:chemistry; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids / Q000032:analysis; D011869:Radioligand Assay; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017421:Sequence Analysis; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; C107608:FABP7 protein, human; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins