Recombinant CMV gB Protein Mutations for Soluble Trimer Production
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CMV vaccine strategies face challenges in producing effective immune responses due to the hydrophobic nature of the cytomegalovirus glycoprotein gB, which causes aggregation and adhesion issues during recombinant expression and purification.
Innovation Solution
Introducing specific mutations into the gB protein to create glycosylation sites and reduce hydrophobicity, such as N-glycosylation sites in hydrophobic surface regions, and adding a heterologous sequence at the C-terminus to mask the hydrophobic surface, facilitating recombinant production and secretion of monomeric trimers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type gB protein is used for recombinant expression, then the protein maintains its natural trimeric structure and immunogenicity, but the hydrophobic surface causes aggregation and adhesion issues that reduce production efficiency and purification ease
Solution Approach 1:
The patent introduces a heterologous sequence as an intermediary component that binds to the hydrophobic surface of the gB protein. This intermediary masks the problematic hydrophobic regions, preventing aggregation and adhesion while allowing the gB protein to maintain its immunogenic trimeric structure. The heterologous sequence acts as a bridge that resolves the conflict between maintaining natural structure and enabling efficient production.
Solution Approach 2:
The patent modifies the physical-chemical parameters of the gB protein by introducing mutations that alter the hydrophobicity of the surface. These parameter changes reduce the tendency of the protein to aggregate and adhere to surfaces, thereby improving production efficiency and purification ease while preserving the essential immunogenic properties.
2Stability of the object's composition
If wild type gB protein is used for recombinant expression, then the protein maintains its natural structure, but adhesion to surfaces and aggregation occur during production and purification
Solution Approach 1:
The heterologous sequence serves as a protective intermediary that binds to the hydrophobic surface of the gB protein. This intermediary layer prevents direct contact between the hydrophobic surfaces of different gB trimers, thereby eliminating aggregation and adhesion to surfaces during production and purification processes, while the underlying gB protein structure remains intact.
Solution Approach 2:
The patent converts the harmful hydrophobic surface property into a beneficial feature by introducing the heterologous sequence. The heterologous sequence specifically binds to the hydrophobic regions, transforming them from aggregation-prone surfaces into controlled interaction interfaces. This converts the harmful adhesion and aggregation into a beneficial masked state that improves production and purification.
3Productivity
If mutations are introduced to reduce hydrophobicity, then production and purification efficiency improve, but the protein structure may be altered
Solution Approach 1:
The patent applies local quality modification by introducing mutations specifically in the hydrophobic surface regions of the gB protein, while leaving the core immunogenic structures unchanged. This localized approach allows the protein to maintain its essential trimeric structure and immunogenicity while improving production and purification efficiency through reduced hydrophobicity in specific areas.
Solution Approach 2:
The patent makes controlled parameter changes by introducing specific mutations that alter the hydrophobicity of surface regions. These parameter changes are carefully designed to improve solubility and reduce aggregation without compromising the overall structural integrity and immunogenicity of the gB protein.
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The invention generally relates to recombinant human cytomegalovirus (CMV) gB proteins and immunogenic fragments thereof, which do not comprise a transmembrane (TM) domain; and comprise one or more mutations that reduce the aggregation between the monomeric trimers of gB, and/or adhesion of the monomeric trimer of gB to the host cell.