On the expression of recombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3) Rosetta strains.

The CRISPR-Cas9 system is a new tool that has been extensively used for genome editing. The system is composed of a Cas9 endonuclease, which has the function of cleaving DNA at a specific site, and a guide RNA (gRNA), which contains the sequence of the cleavage site that is the target of editing. Despite the great interest that has been generated because of the utility of Cas 9 as a molecular tool and a potential therapeutic protein, the production of the 158 kDa recombinant Cas9 protein derived from Streptococcus pyogenes remains a challenge. Here, we systematically evaluated the expression of recombinant Cas9 protein in two different E. coli strains in complex and defined media. The recombinant protein showed improved expression in E. coli BL21(DE3), while only traces of Cas9 protein could be detected in the Rosetta (DE3) strain as a result of much lower mRNA levels. The greatest Cas9 protein expression in defined media containing glucose was observed at an induction temperature of 30 °C and with 8 h of post induction time using IPTG in shake flasks. The protein concentration obtained during a batch bioreactor culture was approximately 420.1 mg/L with 6 h of post induction time. The results demonstrated the possibility of efficient Cas9 protein expression in batch mode using E. coli BL21(DE3) and a simple defined medium and also showed the potential for further improvements that could facilitate large-scale production.

Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.

This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution.