Protein glutaminase (PG) is an enzyme that specifically catalyzes the deamidation of glutamine residues on proteins or peptides, remarkably improving the solubility, emulsification and foaming properties of food proteins and, thereby, conferring great potential in food industry applications. PG is primarily produced from wild strains of Chryseobacterium proteolyticum and the low enzyme production yield restricts large-scale industrial applications. In this context, by evaluating different cleavage site insertions between the pro-region and mature domain of PG as well as different linkers flanking the cleavage site, an E. coli expression and purification protocol has been developed to produce active recombinant PG. To simplify the production workflow, we developed a sequential dual expression system. More than 15 mg of pure and active PG was obtained from 1 L of shaking-flask bacteria culture by one-step nickel affinity chromatography purification. The enzymatic characteristics of the recombinant PG protein were similar to those of native PG. For the deamidation effect of recombinant PG, the deamidation degree (DD) of gliadin reached up to 67% and the solubility increased 84-fold. Thus, this study provides a practical approach to mass producing active PG proteins and investigates its potential applications on food proteins.