Periplasmic synthesis and purification of the human prolactin antagonist ?1-11-G129R-hPRL

Abstract The human prolactin antagonist ? 1-11 -G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of variety cells expressing receptors. Periplasmic expression proteins in E. coli has been considered an option for obtaining soluble and correctly folded protein, as alternative to cytoplasmic production. aim this work was, therefore, synthesize first time, antagonist, testing different activation temperatures purifying it by classical chromatographic techniques. BL21(DE3) strain was transformed plasmid based on pET25b( +) vector, DsbA signal sequence cDNA sequence. Different doses IPTG were added, activating under temperatures, extracting periplasmic fluid via osmotic shock. best conditions achieved at 35 °C 5 h using 0.4 mM IPTG, which gave specific 0.157 ± 0.015 ?g/mL/A 600 final optical density 3.43 0.13 A . Purification carried out nickel-affinity chromatography followed size-exclusion chromatography, quantification being performed high-performance (HPSEC). characterized SDS-PAGE, Western blotting, reversed-phase liquid (RP-HPLC) MALDI-TOF–MS. product presented > 95% purity its antagonistic effects evaluated vitro view potential clinical applications, including inhibition cancer overexpressing receptor antidiabetic properties, taking also advantage fact obtained form without initial methionine.