Mutated PglB Oligosaccharyltransferases for Efficient Saccharide Transfer
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Solution Overview
Problem
Existing PglB oligosaccharyltransferases exhibit varying efficiency in transferring saccharides to proteins, necessitating improved enzymes capable of efficiently catalyzing the transfer of a range of saccharides to proteins containing the required glycosylation motif.
Innovation Solution
Engineered PglB oligosaccharyltransferases with specific amino acid substitutions, such as G477A/S, P195R/C/H, and Y77H, enhance the efficiency of saccharide transfer to proteins, particularly with capsular saccharides of Streptococcus pneumoniae and O-antigens of Shigella sonnei, achieving up to 16-fold yield increases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type PglB oligosaccharyltransferase is used, then the enzyme can transfer saccharides to proteins with the required glycosylation motif, but the transfer efficiency varies and is insufficient for certain saccharide structures
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues in the PglB enzyme (e.g., positions 477, 195, 77, 311, 570) to alter the enzyme's catalytic properties. These point mutations modify the enzyme's active site configuration and substrate binding characteristics, enabling efficient transfer of diverse saccharide structures including capsular polysaccharides and O-antigens that the wild-type enzyme cannot effectively process
Solution Approach 2:
The engineered PglB variants achieve universality by gaining the ability to transfer multiple types of saccharides (capsular polysaccharides, O-antigens, lipopolysaccharides) to various protein substrates containing different glycosylation motifs. The mutated enzyme maintains broad substrate acceptance while significantly improving transfer efficiency across different saccharide-protein combinations
2Ease of manufacture
If chemical modification methods are used to produce glycoconjugate vaccines, then production can proceed with existing technology, but production costs are high and homogeneity is reduced
Solution Approach 1:
The patent replaces chemical modification methods with a biological enzymatic system. The engineered PglB oligosaccharyltransferase catalyzes the transfer of saccharides to proteins in a biologically controlled manner, substituting non-specific chemical reactions with highly specific enzymatic catalysis. This substitution enables precise control over glycosylation sites and outcomes, producing homogeneous glycoconjugates with defined structures
Solution Approach 2:
The engineered PglB enzyme performs self-service by autonomously catalyzing the glycosylation reaction without requiring external chemical reagents or complex purification steps. The enzyme naturally recognizes the glycosylation motif (D/E-Z1-N-Z2-S/T) and transfers saccharides efficiently, enabling a streamlined production process that maintains feasibility while improving product quality
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 engineered PglB enzymes significantly improve the yield of glycosylated proteins by 1.1- to 16,000-fold, enhancing the production of glycoconjugate vaccines and ensuring higher homogeneity and safety.
Implementation Method 1
Engineered PglB oligosaccharyltransferases with specific amino acid substitutions, such as G477A/S, P195R/C/H, and Y77H, enhance the efficiency of saccharide transfer to proteins
Data Source
AI summary
The present disclosure provides mutated PglB oligosaccharyltransferase enzymes, polynucleotides that encode the mutated PglB oligosaccharyltransferase enzymes, host cells capable of expressing the engineered PglB oligosaccharyltransferase enzymes, N-glycosylated proteins that are made using the engineered PglB oligosaccharyltransferase enzymes, and methods of using the engineered PglB oligosaccharyltransferase enzymes.