Bio-better Glucarpidase Glycoengineering Service

Professional Glucarpidase Glycoengineering Service at Creative Biolabs

Glucarpidase is also known as carboxypeptidase G2, which is a protease derived from Escherichia coli. Glucarpidase is usually used to treat acute overdose or poisoning of methotrexate (MTX) or other similar drugs, its mechanism of action is to convert MTX into inactive metabolites, thereby reducing its toxicity in the body. However, its clinical use will be limited by low activity and the production of antibodies after repeated administration. For these reasons, "bio-better" glycoengineering is needed for glucosidase. Creative Biolabs provides specialized glucosidase glycoengineering services. The transformation of glucarpidase through glycoengineering helps to improve its pharmacological properties, enhance its therapeutic effect, reduce adverse reactions, and provide patients with safer and more effective options.

• Recombinantion of glucarpidase

We determine the sugar structure of glucarpidase that needs to be changed according to client needs, such as sugar size, connection method or type, etc. Then we use the glucarpidase gene from Pseudomonas putida as a basic template to construct a glucarpidase mutant library.

• Construction of recombinant expression vector

We amplify the glucarpidase containing the mutated gene, purify the corresponding PCR product, then digest the product with restriction enzymes, and ligate the digested glucarpidase gene into the appropriate expression vector site.

• Transformation of the host system

We transform the expression vector into competent E. coli and screen for functional glucarpidase.

• Culture and expression

We culture the host system under appropriate conditions and induce the expression of recombinant glucarpidase, then collect the cells and proceed with subsequent steps.

• Purification and identification

We purify protein extracts from cells through affinity chromatography technology to obtain recombinant glucarpidase. Moreover, the obtained products are identified through various advanced technical means.

• Activity assessment

We use MTX as a substrate to provide activity assessment for recombinant glucarpidase.

Fig.1 Glucarpidase-based one-stop glycoengineering service.

Published data

Carboxypeptidase G2 (CPG2) is a zinc-dependent protein dimer that hydrolyzes folic acid and its analogs. For example, CPG2 converts MTX to less toxic pteroic acid, resulting in a rapid reduction in MTX levels in serum samples. Therefore, this enzyme is an effective rescue agent for patients who receive high-dose MTX. In addition, CPG2 is also applied in targeted cancer therapy. It can cleave the glutamate part of a variety of prodrugs to release potent nitrogen mustard drugs and is one of the most commonly used enzymes in antibody-directed enzyme prodrug therapy (ADEPT). However, repeated use of CPG2 may induce a strong immune response, limiting its application in therapy. Therefore, it is of great significance to develop a new CPG2 that is not recognized by the immune system. In this study, the authors screened bacteria that produced CPG2 from the soil and then characterized and studied the produced new CPG2 through isolation, cloning, and expression. This new CPG2 had broad application prospects in the future field of cancer treatment.

Fig.2 Activity of recombinant CPG2 on agar plates.¹

Advantages

• Our scientists master advanced glycoengineering technology, and they effectively improve the glycosyl structure of glucarpidase.
• We have experienced professional teams to provide custom glucarpidase glycoengineering services according to client needs.
• We have a professional Glycoengineering Platform and provide high-level glucarpidase glycoengineering service.

Applications

• Glycoengineering improves the drug properties of glucarpidase, such as stability, bioavailability, and immunogenicity, thereby improving therapeutic efficacy, which is particularly important in the treatment of antifolate overdose poisoning.
• Glycoengineering helps improve the performance of glucarpidase, increasing its stability and activity in production.
• Optimizing the glycosyl structure of glucarpidase improves its application in the field of biotechnology, which is helpful for studying related metabolic diseases, protein degradation, and other mechanisms.

Creative Biolabs has developed a comprehensive glycoengineering platform, and with years of rich experience, we provide bio-better Therapeutic Protein Glycoengineering services, such as Glucocerebrosidase and glucarpidase glycoengineering services. Glycoengineering services can optimize the glycosyl structure of proteins to enhance their therapeutic efficacy and biological activity. In addition, the immunogenicity of therapeutic protein is reduced by adjusting the glycosyl structure, thereby reducing the risk of immune reactions in patients. Please contact us if you would like to consult a specific glycoengineering service.

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Reference

1. Rashidi, Fatma B., et al. "Correction: Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment." Plos one 18.11 (2023): e0294885. Distributed under Open Access license CC BY 4.0, without modification.

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