Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine—-serine analogs.

H.S Lu,C.L Clogston,L.O Narhi,L.A Merewether,W.R Pearl,T.C Boone

doi:10.1016/s0021-9258(19)50345-3

Abstract

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.

Highlights

The folding and oxidation of recombinant human species, since they are usually short-lived
The molecule contains a free cysteine at position 17 and two intramolecular disulfide bonds, C y ~ ~ ~ - Canyd sC~~~s ‘ j ~ - C y(1s6~).~The twodisulfide bonds both has overall secondaryand tertiary structures dif- form two small loops which are separated by 21 amino acids
The difficulty in elucidating a proteinfolding pathway lies in measuring the structural properties of intermediate protein formation in the folding of biologically active recombinant hG-CSF (rhG-CSF), we describe the biological and physicochemicalcharacterization of intermediates and analogs made by site-directed mutagenesis at the Cys residues

Summary

RESULTS

Folding Intermediates and Folding Kinetics of rhG-CSF- was performed using[3H]C2-iodoacetic acidT. able 2 lists. RhG-CSF, Intermediates and Analogs-Table 4lists the in Fig. 8B shows the fluorescence spectra at pH 3. Carboxymethylated than that in A), anadpeak at 304 nm, attributable to Tyr, is intermediates I1and I p have only approximately 3 4 % activity present This suggests that the molecule has undergone of the wild type rhG-CSF. Both ~ ~ G - C S F [ C+~ SSer~36~.4,2~] ~a reversible change in conformation so that energy transfer and r h G - c S F [ c y ~+~S~er74] exhibit very low activity Denaturant,and is completely unfolded at 3.5 M GdnHCl

DISCUSSION

(1986) Methods

Folding intermediateb 11