Oligosaccharyltransferase Polypeptide for Bacterial Glycoconjugate Synthesis
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Solution Overview
Problem
The development and production of glycoconjugate vaccines are laborious and costly, requiring multiple steps including polysaccharide glycan purification and chemical coupling to a protein carrier, which limits their immunogenicity and efficiency.
Innovation Solution
Utilization of an oligosaccharyltransferase polypeptide, such as C.sputorum PgIB, which has enhanced glycan transfer activity, along with a carrier polypeptide and a biosynthetic locus, to produce glycoconjugates in bacterial expression systems, thereby facilitating the synthesis and immunogenicity of vaccines.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis methods are used to produce glycoconjugate vaccines, then the production can be completed, but the process becomes laborious and costly requiring multiple purification and coupling steps
Solution Approach 1:
The patent replaces chemical synthesis methods with a biological expression system. Instead of chemically coupling polysaccharides to proteins in multiple steps, the invention uses a bacterial expression system with oligosaccharyltransferase to naturally glycosylate carrier proteins, thereby synthesizing glycoconjugates through biological processes rather than chemical reactions.
Solution Approach 2:
The bacterial expression system performs self-service by autonomously producing glycoconjugates through the endogenous glycosylation pathway. The oligosaccharyltransferase enzyme naturally transfers glycans to carrier proteins expressing appropriate sequons, eliminating the need for external purification and coupling steps that would otherwise be required.
2Productivity
If standard oligosaccharyltransferase is used, then glycan transfer can occur, but the enzyme activity and glycan transfer efficiency are limited
Solution Approach 1:
The patent applies parameter changes by modifying the oligosaccharyltransferase enzyme through site-directed mutagenesis. Specific amino acid residues in the catalytic domain are mutated to alter the enzyme's glycan specificity and enhance its transfer activity, thereby improving both the efficiency and consistency of glycan transfer to carrier proteins.
3Quantity of substance
If high levels of recombinant proteins are expressed, then sufficient glycoconjugate production is achieved, but toxic effects on bacterial cells occur
Solution Approach 1:
The patent implements dynamics by using an inducible expression system where protein expression is tightly controlled. The system remains dormant during bacterial growth and is only activated when needed, allowing the bacteria to reach high cell densities first, then producing glycoconjugates at high levels only after induction, thereby avoiding toxicity during the growth phase.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The use of the modified oligosaccharyltransferase polypeptide with altered glycan specificity and enhanced enzyme activity significantly improves the production and immunogenicity of glycoconjugates, offering a more efficient and cost-effective method for vaccine development.
Implementation Method 1
The oligosaccharyltransferase PglB identified in C. jejuni, the enzyme responsible for the transfer of glycans to protein acceptor proteins
Data Source
Figure 1A~1B
Figure 2~4
AI summary
The disclosure relates to an oligosaccharyltransferase polypeptidesand theiruse in the synthesis of glycoconjugates in bacterial cells; vaccines and immunogenic compositions comprising said glycoconjugates and their use in the prevention and/or treatment of bacterial infection. Bacterial expression system comprising said oligosaccharyltransferasepolypeptides are also disclosed.