An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli
Section snippets
Materials
DNA polymerase, 1-anilino-8-naphthalene sulfonic acid (ANS) and oleic acid were obtained from Sigma (Sydney, NSW, Australia). E. coli strain BL21 Codon Plus (DE3)-RIL was purchased from Stratagene (La Jolla, CA). All other reagents were of the highest purity commercially available.
Cloning of rat FABP
L-FABP cDNA was generated by reverse-transcription of rat intestinal mRNA with the L-FABP-specific primer HS4 (5′-AAAAGGATCCTGTAAAAGAATATGAAATACAGCC-3′). The L-FABP gene fragment was subsequently amplified from the
Expression of the rat L-FABP in E. coli
The cDNA fragment encoding the complete rat L-FABP sequence was cloned into the pTrc99A plasmid downstream of the hybrid trp/lac promoter, Ptrc, to allow for inducible and efficient intracellular expression of rat L-FABP in E. coli (Fig. 1). Induction of expression was verified by Western blotting and was not detectable in uninduced cells (Fig. 2). L-FABP was detected in the soluble fraction of the E. coli extract and was not detectable in the cell wall fraction or media, indicating the protein
Discussion
L-FABP has previously been expressed in E. coli and isolated [9], [18], [19], [20], [21]. However, the published purification protocol yielded a heterogeneous protein mixture containing contamination with an endogenous E. coli protein. Although this protein does not appear to significantly affect the measured affinities of high affinity ligands such as oleic acid and ANS, our interests are in characterizing affinities of much lower affinity ligands. Therefore, it is necessary to have a highly
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