Improving the refolding yield of interleukin-4 through the optimization of local interactions.
Interleukin-4 (IL-4) is a multifunctional cytokine that plays an important role in the regulation of various immune responses. However, the development of IL-4 or IL-4 variants into potential therapeutic drugs is hindered by the low efficiency of the in vitro refolding process of this protein. In this work, we have investigated the improvement of the refolding yield of IL-4 using two different rational design approaches. The first one is based on the so-called inverse hydrophobic effect and involved the replacement of a solvent exposed, non-conserved, hydrophobic residue (W91) by serine. This led to an increase in stability of 1.4 kcal mol(-1) and shifted the midpoint transition temperature (Tm) from 62 to 70 degrees C. The second approach is based on the stabilization of alpha-helices through the introduction of favorable local interactions. This strategy resulted in the following helix sequence for helix C of IL-4, 68ASAAEANRHKQLIRFLKRLDRNLWGLAG95. The mutant protein was stabilized by 0.5 kcal mol(-1), the Tm shifted to 68 degrees C, and a two-fold increase in the refolding yield was consistently observed. Our results make the large-scale production of IL-4 derivatives economically more viable, suggest that a similar approach can be applied to other related proteins, and may represent a general strategy to improve in vitro refolding yields through the selective optimization of the stability of alpha-helices.
Domingues
H
H
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012, Heidelberg, Germany. hdomingu@embl-heidelberg.de
Peters
J
J
Schneider
K H
KH
Apeler
H
H
Sebald
W
W
Oschkinat
H
H
Serrano
L
L
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019791:Guanidine / Q000378:metabolism; D006801:Humans; D015847:Interleukin-4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D010455:Peptides; D015847:Interleukin-4; D019791:Guanidine