Hanspeter Michel

A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli

A versatile plasmid vector was designed to direct the synthesis of recombinant proteins in either one of two forms that will be biotinylated in Escherichia coli with high efficiency at a single, unique site. The protein of interest can be produced with a peptide substrate for E. coli biotin holoenzyme synthetase (BirA) joined directly to its N terminus, or alternatively, as a fusion to the C terminus of a maltose-binding protein domain (MalE) with the peptide substrate on its N terminus. To maximize the yield of biotinylated protein, the vector is designed to express the substrate in a coupled translation arrangement with the enzyme.

Preparation and characterization of albumin conjugates of a truncated peptide YY analogue for half-life extension.

Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.

Affinity purification and characterization of an anti‐PEG IgM

Anti‐PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti‐PEG IgM was observed using the 30 kDa PEG‐derivatized NuGel (single passage). Purified anti‐PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti‐PEG IgM, in solution and adsorbed on 20 kDa PEG‐derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS–PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution‐digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N‐terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti‐PEG IgM fragments were characterized for binding to pegylated interferon alfa‐2a by ELISA. The results from these studies suggest that affinity purified anti‐PEG IgM and fragments can be used as probes in detection assays for PEG functionalized biotherapeutics in pre‐clinical and clinical studies. Copyright

Purification and characterization of a high-molecular-weight form of recombinant human interleukin-2.

During purification of recombinant Interleukin-2 (rIL-2) by reversed-phase HPLC, early fractions are discarded due to the presence of an unidentified form of rIL-2. A procedure has been developed to isolate and purify this unidentified form of rIL-2. The purification process involves two chromatography steps and utilizes a Bakerbond Carboxy-Sulfon (CS) column under two different conditions. This material, designated as a high-molecular-weight form of rIL-2 (HMWrIL-2), exhibits lower mobility during SDS-PAGE and has apI which is approximately one unit less than that of rIL-2, but has similar bioactivity to rIL-2. Structural analysis through enzymatic cleavage, HPLC peptide mapping, mass spectrometry, sequencing, and amino acid composition revealed that the difference between these two proteins is a C-terminal extension of 11 amino acids. This extension could be the result of a nonstandard translation event.

Profile analysis of oligosaccharides from glycoproteins by PMP labeling. Comparison of chemical and enzymatic release methods using RP-HPLC and mass spectrometry

Publisher Summary Chemical or enzymatic methods can be employed to release intact oligosaccharides. Among various electrophoretic and chromatographic methods that give reliable profile results of the released oligosaccharides, reversed-phase (rp) high-performance liquid chromatography (HPLC) offers an advantage. With bovine fetuin as a model system in the chapter, the use of 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling in the routine rp-HPLC profile analysis of glycoproteins has been evaluated. The advantage in this case is, as the system usually uses volatile or salt-free buffer, the recovered samples are suitable for further analysis without additional manipulation. Unfortunately, oligosaccharides lack a hydrophobic domain as well as a chromophore for sensitive detection. To circumvent this disadvantage, it has been demonstrated that oligosaccharides labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) are suitable for rp-HPLC analysis with conventional UV detection. As a result, two analysis kits are now available commercially to perform this method routinely. A kit performs enzymatic release of N-linked oligosaccharides followed by PMP labeling, and a second kit provides chromatographic conditions for separation of these labeled oligosaccharides. With the use of fetuin as a model system, a study is initiated to examine whether the PMP-labeling and rp-HPLC approach is also suitable for analyzing chemically released oligosaccharides. The recovered PMP-oligosaccharide samples from rp-HPLC are also analyzed by MALDI TOF and LC ESI MS.