Daisuke Nohara

The Multiplicity of N-Glycan Structures of Bovine Milk 18 kDa Lactophorin (Milk GlyCAM-1)

Lactophorin is a heat-stable phosphoglycoprotein, also known as milk glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1). Bovine 18 kDa lactophorin was purified by heparin affinity chromatography from cow’s milk whey. Its N-glycans were obtained by proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by in-gel digestion with peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase (PNGase F). The released N-glycans were derivatized with 2-aminopryridine (PA) and analyzed by matrix-assisted laser desorption ionization quadruple ion trap time of flight mass spectrometry (MALDI-QIT-TOF MS). Among the MS analyzed peaks, 15 peaks were found to be N-glycan molecules as detected by MS2 analysis. These glycans consisted of mono-sialylated bi-, tri-, and tetra-antennary complex-type N-glycans carrying Gal-GlcNAc (LacNAc) or GalNAc-GlcNAc (LacdiNAc) with and without core-fucose.

High performance in protease refolding by application of a continuous system using immobilized inhibitor

In the reoxidative refolding of Streptomyces griseus trypsin, which is a serine protease having three S-S bonds per molecule, a synthetic inhibitor immobilized on agarose beads was applied in order to avoid the digestion of non-renatured protease molecules by renatured ones. A semi-continuous refolding system was fabricated by packing such inhibitor-immobilized gels in a glass column and the optimal operating conditions were investigated. Taking account of the effective conditions surveyed in the system, a continuous refolding system was constructed and operated to achieve higher performance. By application of the continuous system, a marked increase in the recovery rate as well as high recovered activity could be accomplished, i.e., the recovery rate obtained was ca. 40 times higher than that in the semi-continuous system. This system was also revealed to be substantially advantageous since it includes not only effective refolding but also separation, purification and enrichment processes in one operation.

Refolding of Fully Reduced Bovine Pancreatic Trypsin

Refolding of bovine pancreatic trypsin was carried out. When starting with denatured S-S intact trypsin, the recovered activity attained was 95-100%. In contrast, the recovered activity after refolding denatured S-S reduced trypsin was considerably low compared with other proteases that have been worked with previously. Such low recovered activity was attributed to the small amount of fully reduced trypsin used as starting material for complete refolding. Taking this into account, a recovered activity of 86% could be achieved when using inhibitor-immobilized gels.

Renaturation of lysozyme with a protein disulfide isomerase chaperone results in enzyme super activity.

When the oxidative refolding of lysozyme (Lyzm) was carried out in the presence of protein disulfide isomerase (PDI) an increased refolding rate and a recovered activity exceeding 100% were reproducibly observed. The origin of this excess activity was investigated by HPLC, SDS-PAGE, and mass spectrometry and assessed using an assay for Lyzm activity. The refolding of Lyzm was achieved through the formation of PDI-Lyzm intermediates and the excess activity was derived from the nascent lysozyme released from these complexes. The released lysozyme exhibited a higher molecular activity than observed for the native protein.