Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli.
Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.
Strandberg
L
L
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Enfors
S O
SO
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D012995:Solubility; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000085:Acetates; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013205:Staphylococcal Protein A; D001616:beta-Galactosidase; D019342:Acetic Acid