Phagemid Vector for Bidirectional Cloning in Phage Display
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Solution Overview
Problem
Current phage display technologies require cloning sequences twice and/or infecting bacterial cells twice to express an amino acid sequence as a fusion protein with either one of two alternative phage coat proteins, which is inefficient and cumbersome.
Innovation Solution
Phagemid vectors are developed for bidirectional cloning of DNA coding for an amino acid sequence to be fused at the N-terminus of either one or both of two functional coat proteins, using a single phagemid vector and a single cloning step, with DIS linkers enabling fusion with either cp3 or cp8 coat proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current phage display technologies are used to express an amino acid sequence as a fusion protein with either one of two alternative phage coat proteins, then the desired protein display is achieved, but the process requires cloning sequences twice and infecting bacterial cells twice, which is inefficient and cumbersome
Solution Approach 1:
The patent merges two separate cloning operations and two separate infection operations into a single integrated process. The phagemid vector contains two multiple cloning sites ( MCS1 and MCS2) that allow insertion of the same DNA sequence in either orientation, enabling expression with either cp3 or cp8 coat proteins through a single cloning step followed by single infection with helper phage. This combining of operations directly resolves the technical contradiction by maintaining versatility while eliminating procedural complexity.
Solution Approach 2:
The phagemid vector is designed with universal functionality to accommodate either coat protein expression pathway. The presence of two MCS sites with compatible restriction sites (SpeI-BglI) allows the same vector to universally accept and express sequences fused to either cp3 or cp8, making the system multi-functional rather than requiring separate specialized vectors for each coat protein option.
2Adaptability or versatility
If current phage display technologies are used to express an amino acid sequence as a fusion protein with either one of two alternative phage coat proteins, then the desired protein display is achieved, but the process is time-consuming due to repeated cloning and transformation steps
Solution Approach 1:
The phagemid vector is pre-configured with two MCS sites containing compatible restriction sites (SpeI at MCS1 and BglI at MCS2) that are positioned and oriented to enable direct insertion of the same DNA fragment in either orientation. This preliminary preparation of the vector with dual-compatible sites eliminates the need for sequential cloning operations, allowing researchers to achieve both display format flexibility and time efficiency through a single ligation and transformation step.
3Quantity of substance
If traditional phage vectors are used, then high display levels are achieved, but the size and cloning strategy of the heterologous sequence are strongly limited
Solution Approach 1:
The patent segments the coat protein expression function from the heterologous sequence insertion function. The phagemid vector contains separate MCS sites positioned within the cp3 and cp8 coding regions, allowing the heterologous sequence to be inserted independently at either location. This segmentation enables flexible cloning strategies with various restriction enzymes while maintaining the ability to achieve high display levels through the helper phage-mediated expression system.
Data Source
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AI summary
The present invention provides novel technologies for producing and screening fusion proteins on the surface of filamentous phage. In particular, a single vector can be used for generating cell and phage libraries containing a given series of protein sequences fused to either one or other of two phage coat proteins. This approach simplifies and improves the efficiency of the subsequent phage display-based selection of protein-binding molecules having therapeutic or diagnostic utility, such as antibodies, peptides, or epitope-binding regions.