Optimization of production and characterization of a recombinant soluble human Cripto‐1 protein inhibiting self‐renewal of cancer stem cells

Abstract

Human Cripto‐1 is a member of the epidermal growth factor (EGF)‐Cripto‐FRL‐1‐Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto‐1 suppresses the self‐renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto‐1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His‐tagged Cripto‐1 protein. A concentrations of 0.2−0.4 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto‐1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto‐1 protein from a Ni‐column in the smallest volume. Cation exchange column chromatography of the Cripto‐1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto‐1 exhibited high affinity to the anti‐ALK‐4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

“…We addressed this issue using a purification strategy that is based on the creation of a fusion Cripto-1 protein with His-tag sequences that are biologically active [ 5 , 64 ] and which can be purified via affinity chromatography. This is a widely accepted strategy for the purification of recombinantly expressed clinically useful proteins [ 5 , 65 , 66 , 67 ]. Other techniques have been applied to recombinantly express and purify His-tag-fused Cripto-1 protein, particularly in non-mammalian expression systems such as E. coli .…”

“…They found that a functional form of soluble Cripto-1 was difficult to obtain because of the 12 cysteine residues in the protein, which impairs the folding process. They developed a special purification process to obtain His-tagged Cripto-1 protein from inclusion bodies under denatured conditions [ 65 ]. The purification process included not only a Ni-NTA column step, but also a CDR-modified cellulose column to remove cellular debris.…”