A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding

Abstract

Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IB's) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urca or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2°) structure of the urea- and GnHCl-solubilised rPGH showed the absence of α-helical content with the majority of the molecule existing in a ‘random coil’ structure. In contrast, the CTAC-solubilised rPGH displayed significant starting 2° structure (10–15% α helix; 30–40% β structure). The three rPGH preparations were refolded in vitro against weak urea, GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCl solubilised IB's. We conclude that the method of solubilisation of IB's and the resultant difference in the starting 2° structure of rPGH, particularly α-helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.