Figure 1
Preparation of recombinant hΔPLP. (A) Scheme for the preparation and amplification of the synthetic hΔPLP DNA template, which is devoid of the hydrophobic putative transmembrane domains of hPLP, represented by the upper triangles, using the PCR overlap extension technique. (B) Alignment of deduced amino acid sequence of hΔPLP with full length native human PLP. The hΔPLP amino acid sequence derived from the DNA sequence of the pRSET/hΔPLP. The dot line in the hΔPLP sequence indicates the positions of the deleted hydrophobic domains. Underlined C and S represent the cysteine to serine substitutions introduced to minimizing incorrect refolding and to increase solubility of the expressed protein. (C) Coomassie Brilliant Blue-stained gel of the expressed and purified hΔPLP from bacteria; lane 1, bacterial host proteins before IPTG induction (20 μg of cellular proteins); lane 2, bacterial proteins after IPTG induction (20 μg cellular proteins; lane 3, hΔPLP (~1.5 μg) purified by metal chelate affinity chromatography on Ni2+-nitrilotriacetic acid agarose.