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Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccine
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Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccine
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Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccine
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Journal Article
Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccine
2021
Overview
•Successful production of a secreted active CRM197 using Pichia pastoris allows for easy downstream processing.•Yields produced are >100 mg L−1, which is equivalent to recombinant production using native Corynebacterium diphtheriae.•Optimized fermentation and purification strategy results in >95% pure recombinant CRM197.•Vi-typhoid antigen vaccine conjugated to recombinantly produced CRM197 has improved efficacy compared to a non-conjugated variant.
CRM197 is a commonly used glycoconjugate carrier that improves the immunogenicity of vaccines, particularly in infants. Despite the advantages of this diphtheria toxoid mutant, low yields, production in inclusion bodies, and the requirement for specific growth conditions have limited the breadth of successful recombinant protein expression platforms available for its expression. We evaluated Pichia pastoris as a production host, using the methanol inducible AOX1 promoter and a modified α-mating factor signal peptide for secretion into the supernatant. Final purified yields >100 mg L−1 culture were achieved when produced in a bioreactor, which is equivalent to the productivity obtained from bioprocesses using the native Corynebacterium diphtheriae host. Recombinant CRM197 was purified to ≥95% homogeneity and showed the expected endonuclease activity. Furthermore, mice immunized with a Salmonella enterica serovar Typhi capsular Vi antigen conjugated to our recombinant CRM197 showed greater than 5-fold increase in immune response. Overall, the results demonstrate that Pichia pastoris is a suitable expression host for the production of high quality CRM197 for vaccine applications.
