Abstract
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.
Highlights
Inclusion body formation during recombinant pro- should provide insight into fundamentalmechanisms of protein expression in bacteriais of both fundamentalin- tein folding and may contribute to an understanding of terest and practical importanceT. o elucidate molecu- amyloid related pathologies such as Alzheimer’s disease [7]
In one set of experiments, temperature-sensitive foldingmutations in the homotrimeric tailspike proteinof the Salmonella phage P22 have been isolated which increase its incorporation into IBs without significantly affecting the protein’s thermodynamic stability [9]. This has led to the suggestion that IB formation by the l a In particular, a Lyse‘ + Val mutation produces sub- taitspike protein, and perhaps by other proteins as well, is stantially more IL- in inclusion bodies than twheild caused by some deficiency in the foldingpathway, such as the type (61 versus 8%)despite generating a protein more thermal lability of a key folding intermediate [10, 11].The thermodynamicallystablethan wild type
Production ofZL-1/3-The plasmid containing the wild type IL-1/3 cDNA engineered from expression in E. coli has been described, along with procedures for cell transformation, growth, and IL-loinduction [28]
Summary
Production ofZL-1/3-The plasmid containing the wild type IL-1/3 cDNA engineered from expression in E. coli has been described, along with procedures for cell transformation, growth, and IL-loinduction [28]. Temperature of aggregation was determined in the above buffer by fluorescence light scattering experiments?Briefly, solutions of 1mg/ ml purified protein in the above buffer were heated in a microcuvette from 25 to 60 "Ca t 0.5 "C/min and agg;egation monitored by apparent light scattering in the SLM-8000 spectrofluorimeter with excitation and emission wavelengths set a t 500 nm. Studies were conducted in reducing agent to suppress aggregationvia disulfidebond formation by the unpaired Cys residues of the protein The effectiveness of this treatment was confirmed by the absence of multimeric forms in nonreducing SDS-PAGE of iodoacetamide-cappedsamples of thermal aggregates (data notshown). Fluorescence yields were converted to the fraction of unfolded protein, FaP (calculated by extrapolation of unfolded and native base lines through the transition zone) and midpoints of unfolding determined [34]. Ferndata were converted to AG values by nonlinear least squares fitting to a two-state model using NLIN (SAS Institute, Cary, NC)
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