Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni(2+)-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.

Human protein-disulfide isomerase (hPDI)-related protein (hPDIR), which we previously cloned from a human placental cDNA library (Hayano, T., and Kikuchi, M. (1995) FEBS Lett. 372, 210 -214), and its mutants were expressed in the Escherichia coli pET system and purified by sequential nickel affinity resin chromatography. Three thioredoxin motifs (CXXC) of purified hPDIR were found to contribute to its isomerase activity with a rank order of CGHC > CPHC > CSMC, although both the isomerase and chaperone activities of this protein were lower than those of hPDI. Screening for hPDIR-binding proteins using a T7 phage display system revealed that ␣ 1 -antitrypsin binds to hPDIR. Surface plasmon resonance experiments demonstrated that ␣ 1 -antitrypsin interacts with hPDIR, but not with hPDI or human P5 (hP5). Interestingly, the rate of oxidative refolding of ␣ 1 -antitrypsin with hPDIR was much higher than with hPDI or hP5. Thus, the substrate specificity of hPDIR differed from that associated with isomerase activity, and the contribution of the CSMC motif to the oxidative refolding of ␣ 1 -antitrypsin was the most definite of the three (CSMC, CGHC, CPHC). Substitution of SM and PH in the CXXC motifs with GH increased isomerase activity and decreased oxidative refolding. In contrast, substitution of GH and PH with SM decreased isomerase activity and increased oxidative refolding. Because CXXC motif mutants lacking isomerase activity retain chaperone activity for the substrate rhodanese, it is clear that, similar to PDI and hP5, the isomerase and chaperone activities of hPDIR are independent. These results suggest that the central dipeptide of the CXXC motif is critical for both redox activity and substrate specificity.Protein-disulfide isomerase (PDI) 1 catalyzes the oxidation, reduction, and isomerization of protein disulfide bonds (1). It is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding that occurs within the luminal space of the endoplasmic reticulum (ER) (2-4). Recently, many PDI homologs have been identified (5-7), and elucidation of their functions, most of which are unknown, may explain the observed diversity of these proteins. PDI has two distinct regions that contain the CGHC sequence. This sequence is similar to a motif found in thioredoxin, CGPC (also called the TX or CXXC motif). CGHC sequences serve as the active sites in PDI for thiol-disulfide bond exchange reactions (8). PDI and its homologs form a diverse protein superfamily whose members are characterized by two or three CXXC motifs (5).We have identified the cDNA of a novel human PDI (hPDI)-related protein (hPDIR) from a human placental cDNA library (9). The deduced amino acid sequence of hPDIR indicates that it contains three CXXC motifs (Cys-Ser-Met-Cys, Cys-Gly-HisCys, and Cys-Pro-His-Cys) and a putative ER retention signal (Lys-Glu-Glu-Leu) at its carboxyl terminus (9). Based on the number and relative positions of CXXC motifs...

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The antibody manufacturing process consists of the cell culture process using animal cells and the purification process including several chromatography steps and filtration steps. The purification process is important to obtain high quality bio-pharmaceutical products by removing impurities, and measurement of antibody concentrations is a fundamental and essential factor to control the manufacturing process and to confirm the yield of each purification step. Here we introduce the optical rotation method as a resolution for measuring antibody concentrations in the manufacturing process. Feasibility evaluation showed that optical rotation is applicable for measuring a wide range of antibody concentrations even in cell culture supernatants containing various contaminants. In addition, as examples of application to the process analytical technology, we show the possibility of the optical rotation method in the continuous cell culture and chromatography process, and the tangential flow filtration concentration process.