The folding pathway of human epidermal growth factor (EGF) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. Oxidative folding of the fully reduced EGF proceeds through 1-disulfide intermediates and accumulates rapidly as a single stable 2-disulfide intermediate (designated as EGF-II), which represents up to more than 85% of the total protein along the folding pathway. Among the five 1-disulfide intermediates that have been structurally characterized, only one is native, and nearly all of them are bridges by neighboring cysteines. Extensive accumulation of EGF-II indicates that it accounts for the major kinetic trap of EGF folding. EGF-II contains two of the three native disulfide bonds of EGF, Cys(14)-Cys(31) and Cys(33)-Cys(42). However, formation of the third native disulfide (Cys(6)-Cys(20)) for EGF-II is slow and does not occur directly. Kinetic analysis reveals that an important route for EGF-II to reach the native structure is via rearrangement pathway through 3-disulfide scrambled isomers. The pathway of EGF-II to attain the native structure differs from that of three major 2-disulfide intermediates of bovine pancreatic trypsin inhibitor (BPTI). The dissimilarities of folding mechanism(s) between EGF, BPTI, and hirudin are discussed in this paper.

Human epidermal growth factor (EGF) contains three disulfides and 53 amino acids. Reduced/denatured EGF refolds spontaneously in vitro to acquire its native structure. The mechanism of this folding process has been elucidated by structural analysis of both acid and iodoacetate trapped intermediates. The results reveal that the folding is accompanied by a sequential flow of unfolded EGF (0-disulfide) through three groups of folding intermediates, namely 1-disulfide, 2-disulfide, and 3-disulfide (scrambled) EGF isomers, to reach the native structure. Equilibrium occurs among isomers of each class of disulfide species, and the composition of intermediates appears to be highly heterogeneous. Together, at least 27 fractions of folding intermediates have been identified, but there exist only limited numbers of well populated species which constitute more than 80% of the total intermediates found during EGF folding. Six species of such well populated intermediates have been isolated, which included two 1-S-S, two 2-S-S, and two 3-S-S scrambled species. Their disulfide structures have been identified here. Both 1-S-S isomers are found to contain non-native disulfides. One of the 2-S-S species consists of two non-native disulfides and the other admits two native disulfides. Among the six disulfides of the two scrambled species, only one is native. Together, native disulfides constitute 25% of the total disulfides found in these six well populated intermediates. These results contrast sharply to those observed with bovine pancreatic trypsin inhibitor, which has shown that well populated folding intermediates consist of exclusively native disulfides (Weissman, J. S., and Kim, P. S. (1991) Science 253, 1386-1393). We propose that well populated folding intermediates, regardless of whether they contain native or non-native disulfides, do not necessarily represent the productive species and specify the folding pathway. Furthermore, conditions influencing the efficiency of EGF folding have been investigated. It is demonstrated here that under optimized compositions of redox agents, including the use of cysteine/cystine and protein disulfide isomerase, the in vitro folding of EGF could be achieved quantitatively within 1 min.

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Fusions between the cloned yeast a-factor structural gene and chemically synthesized DNA segments encoding human protein analogs have been constructed. The gene fusions encode hybrid proteins that include the first 89 amino acids of the native a-factor precursor fused to either a small (j8-endorphin, 31 amino acids) or large (a-interferon, 166 amino acids) foreign protein. Proteolytic cleavage sites involved in a-factor maturation from the native precursor immediately precede the foreign peptide in the hybrid protein.The a-factor promoter was utilized to express the gene fusions in Saccharomyces cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium. The processing of the hybrid proteins has been characterized by amino acid sequence analysis of the secreted proteins. The proteolytic cleavages involved in the maturation of a-factor peptides from the native precursor also occur accurately in the hybrid protein. In addition, cleavages occurred on the carboxyl side of two lysines within the .6-endorphin peptide. Internal cleavages in the interferon protein were also detected. However, in this case, the cleavages occurred at a very low frequency such that >95% of the secreted interferon remained intact.