Recombinant Protein Purification Using Dual Affinity Matrices
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Solution Overview
Problem
The challenge in protein purification is the co-purification of contaminant host cell proteins with the protein of interest due to non-consecutive histidine residues or metal binding motifs, which bind to nickel and cobalt containing purification resins, leading to impure protein preparations.
Innovation Solution
Development of variant host cells where essential proteins are fused with affinity binding tags or mutated to prevent binding to metal chelating matrices, allowing for the use of both metal and non-metal affinity matrices for purification, enabling the separation of recombinant target proteins from contaminants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a histidine tag is used to purify the protein of interest via metal chelating chromatography, then the protein can be efficiently captured and purified, but contaminant host cell proteins containing non-consecutive histidine residues or metal binding motifs will also bind to the resin and co-purify
Solution Approach 1:
The invention extracts and removes contaminant host cell proteins from the system by creating a derivative host cell where these contaminants are fused to affinity tags. This allows selective removal of contaminants through affinity chromatography while the His-tagged protein of interest passes through or is separately purified
Solution Approach 2:
An affinity-tagged version of the contaminant protein serves as an intermediary that binds to the affinity matrix. This intermediary captures other contaminant proteins that would otherwise co-purify with the target protein, acting as a decoy or trap for contaminants
2Productivity
If contaminant proteins are allowed to bind to the metal chelating resin, then the purification process is simpler and faster, but the final protein preparation remains impure
Solution Approach 1:
The purification process is segmented into two distinct steps: first, capture of the His-tagged protein of interest on the metal chelating resin; second, removal of affinity-tagged contaminant proteins on the affinity matrix. This segmentation allows each step to optimize for its specific function while achieving both speed and purity
Solution Approach 2:
The contaminant proteins are pre-modified in the derivative host cell to include affinity tags. This preliminary action ensures that when the cell lysate is applied to the purification system, contaminants are already equipped with the necessary binding moiety for selective removal, eliminating the need for post-purification cleanup steps
3Manufacturing precision
If the host cell genome is modified to fuse essential proteins with affinity binding tags, then contaminant proteins can be selectively removed, but the host cell genetics become more complex
Solution Approach 1:
The invention changes the chemical parameter of the host cell proteins by fusing affinity tags (such as polyhistidine tags or other metal-binding sequences) to essential host cell proteins. This parameter change enables the proteins to bind to metal chelating resins, allowing for their selective removal during purification while maintaining normal host cell function
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
This method effectively isolates recombinant proteins by preventing contaminant proteins from co-purifying, resulting in a purified protein of interest, as demonstrated by the use of chitin and nickel affinity matrices in the described examples.
Implementation Method 1
a metal chelating matrix where the contaminant proteins are washed away and a pure protein is recovered... This binds to a metal containing column... nickel and/or cobalt containing purification resins
Implementation Method 2
at least one of the plurality of essential proteins is additionally fused to an affinity binding tag encoded by the genome, the fusion proteins being capable of binding to a non-metal affinity matrix... an immunoaffinity tag, a peptide tag selected from hemagglutinin, c-myc, T7, Glu-Glu, GST-tag, ZZ, GB1, MCP, and ACP, a streptavidin binding tag or a chitin binding domain tag
Data Source
AI summary
Compositions relating to a combination of two types of separation matrix; and to variant host cells which contain at least one essential host protein that is fused to an affinity binding tag or has been mutated to replace at least two of a plurality of histidines or basic amino acids are provided. Methods are also provided that relate to isolating a recombinant protein from a lysate.