High-yield soluble expression and affinity-tag-free purification of growth hormone receptor antagonist B2036 in Escherichia coli.

Acromegaly is a severe endocrine disease, and growth hormone receptor antagonist B2036 constitutes the core protein backbone of pegvisomant. Conventional B2036 production based on inclusion bodies or periplasmic expression suffers from low yield, complicated refolding, and multi-step purification. Herein, we developed a highly efficient, refolding-free bioprocess for soluble expression of B2036 in Escherichia coli using SUMO fusion technology. The entire fermentation cycle was controlled within 24 h, and fermentation optimization via response surface methodology enabled a high-yield soluble yield of 0.95 g/L. For downstream processing, an industrial-scalable, affinity-tag-free strategy using only two chromatography steps (anion-exchange and mixed-mode chromatography) was established, producing 498.47 mg/L B2036 with 97.12% purity, representing a 20-fold improvement over previous reports. The obtained B2036 showed correct sequence, precise disulfide pairing, and strong biological activity with an IC₅₀ of 0.67 nmol/L. By removing protein refolding and metal-affinity chromatography, this simplified and robust workflow remarkably improves productivity and product quality, offering a cost-effective and scalable technical platform for large-scale manufacturing of pegvisomant biosimilars. Collectively, these findings provide a valuable reference for developing efficient soluble expression and downstream purification processes of other intrachain disulfide-bonded proteins in E. coli.