Charge heterogeneity of insulin fusion proteins expressed in Escherichia coli is not due to proteolytic degradation.
Evaluation of the yield of expression of exogeneous protein in transformed Escherichia coli cells by means of one-dimensional SDS-PAGE often leads to overestimation and miscalculation. For example, it is possible that proteins of similar size comigrate and thus mask the overexpressed product band. Therefore, two-dimensional electrophoresis was used to analyze two types of recombinant fusion proteins, i.e., a beta-galactosidase insulin fusion protein and a interleukin II insulin fusion protein, directly after fermentation. We found that production scale expression products show charge and size heterogeneity. The heterogeneous protein spots were characterized by subsequent blotting onto Immobilon membrane and by N-terminal sequencing. Some of the separated spots were either N-terminally blocked or already degraded to some extent. The integrity of the actual product component of the fusion protein was examined with a C-terminus-specific antibody and by Western blot analysis of the 2D gels.
Hoechst AG, Frankfurt/Main, Germany.
S
S
Müllner
B
B
Karbe-Thönges
D
D
Tripier
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005285:Fermentation; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis; D012967:Sodium Dodecyl Sulfate; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D007328:Insulin; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D001616:beta-Galactosidase; D010447:Peptide Hydrolases