Inclusion body formation and protein stability in sequence variants of interleukin-1 beta.


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. Study holds ProTherm entries: 17, 18, 19, 20, 21, 22, 23, 24 Extra Details: additive : EDTA(2 mM),dG and ddG were measured in the presence of 1.35M GdnHCl human interleukin-1 beta; protein stability; thermal stability;,inclusion body formation; folding intermediate

Submission Details

ID: 9NkveP3j3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:14 p.m.

Version: 1

Publication Details
Chrunyk BA;Evans J;Lillquist J;Young P;Wetzel R,J. Biol. Chem. (1993) Inclusion body formation and protein stability in sequence variants of interleukin-1 beta. PMID:8394358
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2KH2 2009-09-08 Solution structure of a scFv-IL-1B complex
6I1B 1992-10-15 HIGH-RESOLUTION THREE-DIMENSIONAL STRUCTURE OF INTERLEUKIN-1 BETA IN SOLUTION BY THREE-AND FOUR-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
7I1B 1992-10-15 HIGH-RESOLUTION THREE-DIMENSIONAL STRUCTURE OF INTERLEUKIN-1 BETA IN SOLUTION BY THREE-AND FOUR-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
4GAI 2013-02-20 1.49 Crystal structure of EBI-005, a chimera of human IL-1beta and IL-1Ra
2NVH 2006-12-12 1.53 Determination of Solvent Content in Cavities in Interleukin-1 Using Experimentally-Phased Electron Density
1L2H 2003-02-04 1.54 Crystal structure of Interleukin 1-beta F42W/W120F mutant
3POK 2011-01-19 1.7 Interleukin-1-beta LBT L3 Mutant
4G6M 2012-12-19 1.81 Crystal structure of human IL-1beta in complex with therapeutic antibody binding fragment of gevokizumab
1IOB 1996-08-17 2.0 INTERLEUKIN-1 BETA FROM JOINT X-RAY AND NMR REFINEMENT
1I1B 1990-01-15 2.0 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN INTERLEUKIN-1BETA AT 2.0 ANGSTROMS RESOLUTION
4I1B 1990-04-15 2.0 FUNCTIONAL IMPLICATIONS OF INTERLEUKIN-1BETA BASED ON THE THREE-DIMENSIONAL STRUCTURE
2I1B 1990-04-15 2.0 CRYSTALLOGRAPHIC REFINEMENT OF INTERLEUKIN-1 BETA AT 2.0 ANGSTROMS RESOLUTION
4G6J 2012-12-19 2.03 Crystal structure of human IL-1beta in complex with the therapeutic antibody binding fragment of canakinumab
3LTQ 2011-02-16 2.1 Structure of Interleukin 1B solved by SAD using an inserted Lanthanide Binding Tag
1TWE 2004-12-07 2.1 INTERLEUKIN 1 BETA MUTANT F101Y
1TOO 2004-12-07 2.1 Interleukin 1B Mutant F146W
5I1B 1992-01-15 2.1 A COMPARISON OF THE HIGH RESOLUTION STRUCTURES OF HUMAN AND MURINE INTERLEUKIN-1B
1T4Q 2004-12-07 2.1 Interleukin 1 beta F101W
5MVZ 2017-02-15 2.15 Fab 4AB007 bound to Interleukin-1-beta
4GAF 2013-02-20 2.15 Crystal structure of EBI-005, a chimera of human IL-1beta and IL-1Ra, bound to human Interleukin-1 receptor type 1
1TP0 2005-02-08 2.2 Triple mutation in interleukin 1 beta cavity:replacement of phenylalanines with tryptophan.
5BVP 2015-09-02 2.2 The molecular mode of action and species specificity of canakinumab, a human monoclonal antibody neutralizing IL-1beta
1TWM 2004-12-07 2.26 Interleukin-1 Beta Mutant F146Y
9ILB 1999-01-06 2.28 HUMAN INTERLEUKIN-1 BETA
1S0L 2004-03-30 2.34 Interleukin 1 beta mutant F42W
1HIB 1994-01-31 2.4 THE STRUCTURE OF AN INTERLEUKIN-1 BETA MUTANT WITH REDUCED BIOACTIVITY SHOWS MULTIPLE SUBTLE CHANGES IN CONFORMATION THAT AFFECT PROTEIN-PROTEIN RECOGNITION
1ITB 1998-02-04 2.5 TYPE-1 INTERLEUKIN-1 RECEPTOR COMPLEXED WITH INTERLEUKIN-1 BETA
4DEP 2012-03-21 3.1 Structure of the IL-1b signaling complex
3O4O 2010-09-01 3.3 Crystal structure of an Interleukin-1 receptor complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.8 Interleukin-1 beta P46648 IL1B_CERAT
95.4 Interleukin-1 beta P79182 IL1B_MACFA
95.4 Interleukin-1 beta P51493 IL1B_MACNE
96.1 Interleukin-1 beta P48090 IL1B_MACMU
100.0 Interleukin-1 beta P01584 IL1B_HUMAN