Inclusion body formation and protein stability in sequence variants of interleukin-1 beta.
Inclusion body formation during recombinant protein expression in bacteria is of both fundamental interest and practical importance. To elucidate molecular mechanisms of this process, we are examining the in vitro folding and stability properties of a series of human interleukin-1 beta (IL-1 beta) sequence variants which exhibit widely differing tendencies to form inclusion bodies. Of 67 variants surveyed, nine, including wild type, were purified and their in vitro stability properties determined. One of these, a high inclusion body mutant, exhibited very low solubility in native buffer after purification and was not pursued further. For the other eight sequence variants, no strong correlations were observed between extent of inclusion body formation and either thermodynamic or thermal stability. In particular, a Lys97-->Val mutation produces substantially more IL-1 beta in inclusion bodies than the wild type (61 versus 8%) despite generating a protein more thermodynamically stable than wild type. Furthermore, the Lys97-->Val mutant forms substantial levels of inclusion bodies at 32 degrees C but requires incubation at temperatures greater than 48 degrees C for thermally induced aggregation in vitro. This and other data suggest that the tendency of at least some IL-1 beta variants to form inclusion bodies is most likely related to the stability or solubility of folding intermediates rather than native states. Implications of the structural locations of these mutations are also discussed.
Chrunyk
B A
BA
Macromolecular Sciences Department, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Evans
J
J
Lillquist
J
J
Young
P
P
Wetzel
R
R
D000595:Amino Acid Sequence; D002151:Calorimetry; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D014644:Genetic Variation; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D007375:Interleukin-1; D011994:Recombinant Proteins; D014364:Tryptophan