Fusion Protein for Detection Using Mutant BAP and Protein G
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Solution Overview
Problem
Current protein detection methods, such as ELISA, face limitations in versatility, sensitivity, and stability, particularly with alkaline phosphatases like CIAP and BAP, which suffer from high production costs, thermal instability, and low activity at low concentrations.
Innovation Solution
A fusion protein is developed by combining a protein domain including a C1, C2, or C3 domain of protein G with double mutants of Escherichia coli alkaline phosphatase (BAP), specifically D153G/D330N, D153H/D330N, or K328R/D330N, to enhance versatility, sensitivity, and stability for protein detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CIAP purified from calf small intestines is used, then high detection sensitivity is achieved, but production cost is high and stable quality is difficult to achieve
Solution Approach 1:
The patent uses BAP produced by gene recombination in E. coli as a cost-effective alternative to expensive CIAP purified from calf intestines. The BAP system allows for inexpensive large-scale production while maintaining detection functionality, directly addressing the high production cost issue.
Solution Approach 2:
The patent introduces specific mutations (D153G, D153H, K328R at position 153; D330N at position 330) in BAP to enhance its properties. These parameter changes in the amino acid sequence improve thermal stability and maintain activity at low concentrations, resolving the quality stability issue while keeping production costs low.
2Ease of manufacture
If BAP produced by gene recombination in yeast is used, then production cost is reduced, but excessive glycosylation occurs causing background and viscosity problems
Solution Approach 1:
The patent changes the host organism parameter from yeast to E. coli for BAP production. This parameter change eliminates the excessive glycosylation problem inherent in yeast production, as E. coli does not perform O-glycosylation. The mutations D153G/D330N or D153H/D330N further optimize the enzyme to prevent aggregation and reduce viscosity issues.
Solution Approach 2:
The patent uses E. coli as a disposable production system that can be easily cultured and harvested. This system provides a clean, glycosylation-free BAP product that can be produced at low cost without the background and viscosity problems associated with yeast-produced enzymes.
3Power
If CIAP is used, then high activity is achieved, but thermal stability is poor and maintaining activity for long periods is difficult
Solution Approach 1:
The patent introduces specific amino acid substitutions (D153G, D153H, or K328R at position 153 combined with D330N at position 330) in BAP to enhance thermal stability. These parameter changes in the protein structure allow the enzyme to maintain its active conformation at higher temperatures and over longer periods, directly resolving the thermal stability issue while preserving high enzymatic activity.
4Adaptability or versatility
If enzyme-labeled protein G is used, then versatility is improved and separate antibodies need not be prepared, but detection sensitivity is low and thermal stability is poor
Solution Approach 1:
The patent merges the high antibody-binding capability of protein G (C1, C2, or C3 domain) with the high enzymatic activity and improved thermal stability of mutant BAP. This fusion creates a single molecule that combines the versatility of protein G with the superior detection performance of engineered BAP, resolving both the sensitivity and stability issues.
Solution Approach 2:
The patent creates a composite fusion protein structure where the protein G domain provides antibody binding functionality and the mutant BAP domain provides enzymatic activity with enhanced stability. This composite structure integrates the advantages of both components while eliminating their individual weaknesses, achieving high versatility, sensitivity, and thermal stability simultaneously.
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 fusion protein exhibits superior versatility, high detection sensitivity, and stability, allowing for efficient protein detection across various targets without the need for multiple secondary antibodies and maintaining activity at high temperatures.
Implementation Method 1
a primary antibody for a target material (antigen) adsorbed to the surface of a solid phase is bound via an antigen-antibody reaction
Implementation Method 2
an enzyme-labeled secondary antibody is then added and bound via a second antigen-antibody reaction... when a chromogenic substrate is added, a color reaction occurs
Data Source
AI summary
A fusion protein for protein detection in which are fused: a protein domain including at least one among a C1 domain of protein G, a C2 domain of protein G, and a C3 domain of protein G; and a double mutant D153G/D330N of Escherichia coli alkaline phosphatase (BAP) in which the 153 amino acid residue Asp has been substituted by Gly and the 330 amino acid residue Asp has been substituted by Asn, a double mutant D153H/D330N of Escherichia coli alkaline phosphatase (BAP) in which the 153 amino acid residue Asp has been substituted by His and the 330 amino acid residue Asp has been substituted by Asn, or a double mutant K328R/D330N of Escherichia coli alkaline phosphatase (BAP) in which the 328 amino acid residue Lys has been substituted by Arg and the 330 amino acid residue Asp has been substituted by Asn.