Development of a Highly Efficient System for the Production of the Holoform of Recombinant Cytoglobin

Abstract

Objective: A highly efficient production system for recombinant human cytoglobin using minimal medium has been developed and optimized. This system enables the production of the water-soluble holoform of the protein—in both unlabeled and isotopically labeled forms—in quantities sufficient for comprehensive studies. Methods: The development of the E. coli expression system for the recombinant CYGB gene was performed using the pET-17b plasmid vector. A series of experiments involving multifactorial optimization of CYGB expression conditions was performed, varying incubation temperature and duration, final concentrations of IPTG and δ-ALA, and culture medium composition. Four E. coli strains were tested: SHuffle T7, BL21(DE3)pLysS, JM-109, and BL21(DE3). Expression in rich medium was carried out in TB medium with IPTG at 0.1–0.5 mM; the highest yields were obtained with BL21(DE3)pLysS in TB medium supplemented with 0.2 mM IPTG at 37°C for 20 h. Isolation and purification of the Cygb apoprotein from inclusion bodies under denaturing conditions was performed using 6 M guanidine hydrochloride or urea, followed by renaturation in the presence of hemin. For the water-soluble holoprotein, a system for Cygb production in M9 minimal medium was developed. Cultivation parameters included BL21(DE3)pLysS, IPTG up to 0.15 mM, δ-ALA at 0.8 mM, and post-induction incubation for 16–17 h at 37°C under reduced aeration (180 rpm). For the ¹⁵N-labeled variant, ¹⁵N-ammonium chloride was supplied as the sole nitrogen source. Isolation and purification of the native Cygb holoprotein included homogenization, ammonium sulfate fractionation up to 60% saturation, anion-exchange chromatography on Mono Q-20, concentration by ammonium sulfate precipitation up to 80% saturation, size-exclusion chromatography on Sephadex G-75, and lyophilization. Physicochemical characterization was performed using UV-visible, CD, and NMR spectroscopy, dynamic light scattering, and Raman spectroscopy with 532 nm laser excitation. Results and Discussion: UV-visible, CD, and NMR spectroscopy data indicate that the recombinant cytoglobin is a structured holoform of an alpha-helical protein with a hydrodynamic diameter of approximately 5.4 nm (as determined by dynamic light scattering). It was demonstrated by Raman spectroscopy with 532 nm laser excitation that the heme in both the ferrous and ferric cytoglobin forms exhibits characteristic maxima typical of b-type hemes, with the iron atom being six-coordinated. Preparative quantities of recombinant holo-cytoglobin and its ¹⁵N-labeled variant were obtained using the developed production system. Quality control of the ¹⁵N-labeled cytoglobin by NMR spectroscopy revealed a high degree of ¹⁵N-isotope incorporation into the protein. Furthermore, the ¹H-¹⁵N HSQC spectrum showed broad chemical shift dispersion with no signs of protein denaturation. Conclusions: The obtained results serve as a basis for detailed structural-functional studies of human cytoglobin.