Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design,Journal of Agricultural and Food Chemistry

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Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2024-04-24 , DOI: 10.1021/acs.jafc.4c01406
Xiaodi Li ₁ , Kashif Rahim ₁ , Xingyu Shen ₁ , Xin Cui ₁ , Chao Du ₁ , Guimin Zhang ₁

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Cytotoxic enzymes often exist as zymogens containing prodomains to keep them in an inactive state. Protein-glutaminase (PG), which can enhance various functional characteristics of food proteins, is an enzyme containing pro-PG and mature-PG (mPG). However, poor activity and stability limit its application while tedious purification and activation steps limit its high-throughput engineering. Here, based on structural analysis, we replaced the linker sequence between pro-PG and mPG with the HRV3C protease recognition sequence and then coexpressed it with HRV3C protease in Escherichia coli to develop an efficient one-step purification and activation method for PG. We then used this method to obtain several mutants designed by a combination of computer-aided approach and beneficial point mutations. The specific activity (131.6 U/mg) of the best variant D1 was 4.14-fold that of the wild type, and t_1/2 and T₅₀¹⁰ increased by 13 min and 7 °C, respectively. D1 could effectively improve the solubility and emulsification of wheat proteins, more than twice the effect of the wild type. We also discussed the mechanism underlying the improved properties of D1. In summary, we not only provide a universal one-step purification and activation method to facilitate zymogen engineering but also obtain an excellent PG mutant.

更新日期：2024-04-24

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