Stephen Bayne

In vivo processing of N‐terminal methionine in E. coli

The processing of amino-terminal methionine from cytosolic proteins in E. coli has been investigated in vivo, using amino-terminal-extended human growth hormone (hGH) as a model system. Twenty different hGH-genes with the sequence Met-Xxx-Glu-Glu-hGH where Xxx denotes each of the 20 different amino acids, were constructed and expressed in E. coli. Following purification of the products, the N-terminal amino acid sequences (10 cycles) were determined. The results demonstrate that the removal of methionine is dependent on the amino acid adjacent to methionine, and that the processing is strongly correlated to the radius of gyration of this amino acid. In addition, measurement of the hGH expression level from the 20 clones demonstrated that the small difference in the amino acid extension leads to a change in the specific hGH expression rate.

Cloning and expression of an interleukin-1β precursor and its conversion to interleukin-1β

A gene coding for a N‐terminal precursor of interleukin‐1β (IL‐1β) was cloned and expressed in E. coli. The isolated Met‐Glu‐Ala‐Glu‐IL‐1β precursor was enzymatically converted to IL‐1β by means of dipeptidylaminopeptidase (DAP I). This method ensured a correct N‐terminal residue and the often observed expression of Met‐IL‐1β was thus avoided. The pure and physically homogeneous product exhibited the characteristic properties of natural IL‐1β. The in vitro biological activity was measured in the lymphocyte‐activating factor assay and was compared to that of natural IL‐1β isolated from stimulated monocyte culture using exactly the same purification procedure. The specific biological activity of both products was 2 × 10−8 U/mg indicating that the recombinant product exhibits full biological activity.

Analysis of the site-specific asparagine-linked glycosylation of recombinant human coagulation factor VLLa by glycosidase digestions, liquid chromatography, and mass spectrometry

The two asparagine-linked glycosylation sites of recombinant coagulation factor VIIa have been characterized by glycosidase digestions, size-exclusion chromatography (SEC), and mass spectrometry (MS). Nine structures were characterized as core fucosylated bi- and triantennary structures with 0-3 sialic-acid residues, which were α2-3 linked to galactose exclusively. Three of the structures had one or two galactose residues substituted by N-acetylgalactosamine. Significant differences were found between the oligosac-charide profiles for the two glycosylation sites in rFVIIa. At Asn322, the degree of sialylation was lower and higher amounts of structures containing N-acetylgalactosamine were found compared to Asn l45.