Figure 1

Cloning of rat Ap-B coding region, expression and purification of the E. coli recombinant rat Ap-B protein. (A) Schematic representation of the pIVEX2.4-Ap-B vector. A 1965 bp-long DNA fragment (Aminopeptidase B) containing the rat Ap-B CDS [18] was cloned into the pIVEX2.4 expression vector. T7 promotor, Ribosome Binding Site (RBS), translation initiation codon (ATG), Histidine tag (His Tag), Factor Xa restriction protease cleavage site (Xa or Factor Xa), NcoI and BamHI restriction sites, stop codon (***), and transcription T7 terminator (T7 Terminator) are indicated. (B) Western blot analysis of the expression of the recombinant rAp-B in E. coli. Lane 1 contains a purified recombinant rAp-B produced in a baculovirus expression system ([29]). Lane 2 contains a crude protein extract from pIVEX2.4-Ap-B transformed cells. Each sample (2 μL) was diluted in 20 μL of 1× Laemli buffer to avoid buffer effects during electrophoresis. Arrows indicate immunoreactive proteins corresponding to the baculovirus produced rAp-B (rAp-B; 650 amino acid residues; [29]) and the E. coli expressed rAp-B (His-rAp-B; 673 amino acid residues), respectively. (C) Purification using Ni-NTA column. The different protein extracts analyzed are the following: C, rAp-B purified from baculovirus expression system used as a positive control; CE, crude protein extract from pIVEX2.4-Ap-B transformed BLi5 cells (5 μL); FT, proteins released after passage through the Ni-NTA column (20 μL); W1 and W2, proteins released after the first and second washing of the Ni-NTA column, respectively (20 μL); E1 and E2, proteins collected after the first and second elution step, respectively (20 μL). (D) Purification of recombinant rat Ap-B using DEAE ion exchange column. Different fractions (43, 45, 47, 49, 51, 53) collected from the ion exchange column and containing rAp-B activity were analysed (20 μL per fraction). (C, D) Samples were run on SDS-PAGE and either stained with silver salts (upper panel) or transferred to a nitrocellulose membrane for western blotting (lower panel). Arrows indicate the E. coli expressed rAp-B (rAp-B). (B, C, D) Relative molecular masses are indicated in kilo Daltons (MM, kDa).