Proteolysis of fusion proteins: stabilization and destabilization of staphylococcal protein A and Escherichia coli beta-galactosidase.
The product yield of staphylococcal Protein A reached only 1.8% of the cell dry weight, while the corresponding value was 14% for a fusion protein composed of Protein A and Escherichia coli beta-galactosidase [1], when produced in the same E. coli host strain, with the same promoter and under identical process conditions. Measurement of the stability of Protein A in vivo showed that it was quickly degraded in the cell with a half-life of 30 min when the protein was expressed alone, but after fusion to beta-galactosidase, the Protein A part became considerably stabilized. In spite of the fast intracellular proteolysis of Protein A, few degradation products could be identified on Coomassie Brilliant Blue-stained SDS/PAGE gels after IgG purification, indicating an even faster degradation of the Protein A fragments. Such degradation products, however, accumulated during incubation of the disintegrated cells. Intracellular degradation intermediates could be demonstrated with the more sensitive Western-blot technique. This technique also revealed that a slow degradation took place not only in the Protein A moiety of the fusion protein, but also in the beta-galactosidase moiety. A control with native beta-galactosidase also showed a weak in vivo proteolysis of this molecule, but it was more stable in free form than in the fused form. This means that the proteolytically very sensitive Protein A was stabilized by fusion with beta-galactosidase, but the originally rather stable beta-galactosidase became slightly more susceptible to proteolysis after the fusion.
Yang
S
S
Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Veide
A
A
Enfors
S O
SO
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D001616:beta-Galactosidase