Improving the refolding yield of interleukin-4 through the optimization oflocal interactions

Interleukin-4 (IL-4) is a multifunctional cytokine that plays an important role in the regulation of various immune responses. However, the developmen t 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 th is 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 midpoin t transition temperature (T-m) from 62 to 70 degreesC. The second approach is based on the stabilization of a-helices through the introduction of favo rable local interactions. This strategy resulted in the following helix seq uence for helix C of IL-4, 68ASAAEANRHKQLIRFLKRLDRNLWGLAG95. The mutant pro tein was stabilized by 0.5 kcal mol(-1), the T-m shifted to 68 degreesC, an d a two-fold increase in the refolding yield was consistently observed. Our results make the large-scale production of IL-4 derivatives economically m ore viable, suggest that a similar approach can be applied to other related proteins, and may represent a general strategy to improve in vitro refoldi ng yields through the selective optimization of the stability of alpha -hel ices. (C) 2000 Elsevier Science B.V. All rights reserved.