EXPRESSION OF MAIZE GAMMA-ZEIN C-TERMINUS IN ESCHERICHIA-COLI

Prolamins containing a highly conserved cysteine-rich C-terminal domai n have been poorly expressed as soluble protein in model systems such as Escherichia coli. Possible reasons have included a combination of t he reducing environment of the bacterial cytoplasm and protein seconda ry structure, Using a bacterial thioredoxin fusion expression system, full-length native gamma zein, native gamma zein C-terminus, and modi fled gamma zein C-terminus, containing 13 amino acid changes, were fou nd to accumulate up to 58, 50, and 42% of the total cellular protein, respectively. The native gamma zein C-terminus fusion protein was six times more soluble (70%) than the full-length fusion protein (12%), fo ur times more soluble than the N-terminus (19%), and eight times more soluble than the modified C-terminus (9%). The modified C-terminal dom ain contained amino acid changes that improved the lysine, isoleucine, and tryptophan content, while removing two evolutionarily conserved c ysteines and one nonconserved cysteine, Expression of the native C-ter minal domain without thioredoxin resulted in decreased solubility (13% ) and decreased expression (8%). In contrast, coexpression with thiore doxin resulted in a sevenfold increase in solubility (86%). These resu lts suggest that insolubility of full-length gamma zein results from s tructural interactions of the N-terminus and that solubility of the C- terminal domain is dependent on proper disulfide bond formation. The a bility to express the C-terminal domain of gamma zein as soluble prote in should allow future identification of important structural elements in gamma zein and similar proteins. (C) 1998 Academic Press.