Yeast Vector for In Vivo Biotinylation of Secreted Proteins
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Solution Overview
Problem
Current methods for producing high-affinity antibodies for biomarker validation are slow and costly, and existing in vivo biotinylation protocols are not compatible with high-throughput approaches, particularly for proteins like CA125, which lacks a mouse antibody, impairing ovarian cancer studies.
Innovation Solution
A vector system for yeast that includes a nucleic acid sequence encoding a biotin acceptor site for golgi-localized biotin ligase, allowing for the expression of secreted, biotinylated recombinant polypeptides, enabling efficient generation of antibodies with high affinity and specificity, including those targeting ovarian cancer biomarkers like CA125.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If chemical biotinylation is used to label recombinant proteins, then biotinylation can be achieved, but lysine residues critical for protein function and conformation are inactivated
Solution Approach 1:
The patent introduces a biotin acceptor site (BAS) as an intermediary component that specifically binds biotinylated ligases without affecting other lysine residues. This mediator enables selective biotinylation at the BAS while preserving the functional integrity of the protein, resolving the contradiction between achieving biotinylation and maintaining protein function
Solution Approach 2:
The invention implements localized biotinylation by designing a specific biotin acceptor site at a defined location on the protein (N- or C-terminus). This local modification approach ensures that only the designated site is biotinylated, while other critical lysine residues remain unaffected, thus maintaining protein function and conformation
2Reliability
If existing in vivo biotinylation protocols are used, then specific biotinylation can be achieved, but the protocols are not compatible with high-throughput approaches
Solution Approach 1:
The patent creates a universal vector system that can accommodate multiple different polypeptide inserts while maintaining the same biotinylation mechanism. The vector contains a biotin acceptor site and biotinylated ligase that work together in a standardized platform, enabling high-throughput production of various biotinylated proteins without requiring protocol optimization for each case
Solution Approach 2:
The invention divides the biotinylation system into separate functional modules: a vector component containing the biotin acceptor site and a ligase component. This segmentation allows independent optimization and assembly of components, facilitating high-throughput workflows where different polypeptide inserts can be independently cloned and expressed in the standardized vector system
3Adaptability or versatility
If yeast display scFv are converted into soluble and labeled recombinant antibodies, then antibodies for immunoassays can be generated, but the process is slow and costly
Solution Approach 1:
The patent incorporates the biotin acceptor site and biotinylated ligase into the yeast display vector system in advance. This preliminary setup allows scFv libraries to be displayed on yeast surface and simultaneously biotinylated during the display process, eliminating the need for separate conversion and labeling steps, thus reducing time and cost while maintaining antibody functionality
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
Facilitates the rapid and cost-effective production of high-affinity, biotinylated antibodies that can be used for biomarker validation and detection, eliminating the need for purification and chemical biotinylation, and allows for the generation of antibodies that inhibit CA125/mesothelin-dependent cell attachment.
Implementation Method 1
a biotin ligase, wherein the secreted recombinant polypeptide and the biotin ligase are localized to the golgi of the yeast cell
Data Source
AI summary
The present invention provides compositions and methods for generating in vivo biotinylated, secreted recombinant polypeptides comprising recombinantly co-expressing in a yeast cell (a) a secreted recombinant polypeptide comprising a biotin accepting site; and (b) a biotin ligase fused to the golgi localization domain so that the biotin ligase is localized to the golgi of the yeast cell where it acts on the biotin-accepting site of the secreted polypeptide as it transits the secretory system.