The influence of energy sources on the proteolysis of a recombinant staphylococcal protein A in Escherichia coli.
The kinetics of proteolysis of a recombinant staphylococcal protein A in Escherichia coli were studied by a Western-blotting-based method. The proteolysis constants obtained from this method are very close to those obtained from the traditional radioactive pulse-chase technique. Protein A was selectively degraded to a great extent, while host proteins were quite stable after heat induction of protein A expression. The proteolysis of protein A was much faster in the presence of energy sources compared to when cells were starved of energy. The degradation rate constants are 2.8 h-1 in the presence of 10 g/l glucose and about 0.4 h-1 in the absence of any external carbon source. The supplementation of glucose to the medium at 0-100 mg/l caused a gradual increase of proteolysis of protein A, but the proteolysis was saturated when the concentration of glucose exceeded 200 mg/l at a cell concentration of about 0.36 g/l. The respiration inhibitor sodium azide completely inhibited the degradation of protein A in glucose-free salt medium but had almost no effect in the presence of glucose. Therefore, the proteolysis process is energy dependent but the energy supply rate obtained by fermentation of glucose is enough to meet this requirement. The proteolysis rate increased with the temperature in the interval 5-45 degrees C but was then reduced due to damage of the proteolysis system by high temperature. At 60 degrees C, the proteolysis ceased completely within 30 min.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
S
S
Yang
S O
SO
Enfors
D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D003470:Culture Media; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A