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 (lB's) using either 6 M guanidinium hydroehloride (GnHCI). 7.5 M urea or by a novel method using a cationic surfactant, cetyltrimeth)'lal~monium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2 °) structure of the urea-and GnHCl-sol ubilised rPG H showed the absence of,r-helical content with the majority of the molecule existing in a "random coil" structure. In contrast, the CTAC-solabilised rPGH displayed significant starting 2 ° structure (10-15% ~ helix; 30-40% fl structure). The three rPGH preparations were refolded in vitro against weak urea, GnHCI or aqueous buffers, resulting in an average refolding ¢ffieiel~cy of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCI 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 ,r.helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.

Solubilization of Recombinant Ovine Growth Hormone with Retention of Native-like Secondary Structure and Its Refolding from the Inclusion Bodies of Escherichia coli

“…More than 90% of the r‐oGH from the inclusion body was solubilized at pH 12 in the presence of 2 M urea with high initial protein concentration (2.6 mg/mL). Solubilization of an inclusion body protein with retention of its secondary structure has been reported by the use of cationic surfactant (33, 34). However, the use of surfactants for solubilization of inclusion body protein necessitated a complex washing procedures to remove the surfactant, resulting in considerable loss of protein.…”