Enhanced Recombinant Protein Production of Soluble, Highly Active and Immobilizable PNGase F

Abstract

High resolution analysis of N-glycans can be performed after their endoglycosidase mediated removal from proteins. N-glycosidase F peptide (PNGase F) is one the most frequently used enzyme for this purpose. Because of the significant demand for PNGase F both in basic and applied research, rapid and inexpensive methods are of great demand for its large-scale production, preferably in immobilizable form to solid supports or surfaces. In this paper, we report on the high-yield production of N-terminal 6His-PNGase F enzyme in a bacterial Escherichia coli SHuffle expression system. The activity profile of the generated enzyme was compared to commercially available PNGase F enzymes, featuring higher activity for the former. The method described here is thus suitable for the cost-effective production of PNGase F in an active, immobilizable form.

Keywords: Capillary electrophoresis; N-glycan; PNGase F enzyme activity; SHuffle cells.

Fig. 1

Comparison of N-glycan profiles of hIgG1 sample digested by 6His-PNGase F (denoted as 6His) and Asparia Glycomics 500 IUB activity PNGase F (AG) enzymes. The insets represent the integrated areas of four major peaks with the corresponding standard deviation from three parallel measurements. Separation conditions: 30 cm total length, 20 cm effective length BFS capillary. HR-NCHO separation gel buffer. Separation voltage: 30 kV in reverse polarity mode. Capillary temperature: 25 °C. Injection sequence: (1) 1.0 psi for 5.0 s HPLC grade water and (2) 2.0 kV/2.0 s sample