Fusion Protein Purification
Fusion Protein Purification - Related Videos
11:22
GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
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Cited by 18 •
2013
In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.
07:35
Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli
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Cited by 48 •
2014
Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.
10:32
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
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Cited by 7 •
2012
A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.
07:17
Purification of Hsp104, a Protein Disaggregase
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Cited by 24 •
2011
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
09:27
Generation of Fluorescent Protein Fusions in Candida Species
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Cited by 6 •
2017
PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.
FAQs
The affinity tag or partner domain provides the feature recognized by a complementary ligand on the chromatography resin. This interaction allows the fusion protein to remain associated with the resin while other components in the biological sample pass through or are removed during washing. Tag–ligand compatibility therefore determines whether capture is selective and effective.
Expression and solubility directly influence how much fusion protein is available for purification and whether it can interact with the resin. Poor solubility can reduce the recoverable fraction, while unsuitable expression can limit yield. Optimizing these properties before chromatography helps the binding step perform consistently and supports better overall purity and recovery.
Elution conditions weaken the interaction between the tagged protein and its resin ligand. Adjusting buffer pH or imidazole concentration can promote release after unwanted components have been washed away. The selected condition must separate release from earlier capture, because premature weakening can reduce binding, whereas insufficient change can leave protein associated with the resin.
A typical workflow begins with a biological sample containing the recombinant fusion protein, followed by contact with an affinity resin. The sample is applied so the tagged protein can bind, unbound material is removed by washing, and buffer conditions are then changed to elute the retained protein. Expression, solubility, binding, and elution conditions are optimized across the workflow.
This approach is useful when researchers need isolated recombinant protein for downstream biological investigations. The purified material can support structural studies, enzyme assays, antibody generation, and functional research. Its value comes from connecting selective isolation with experiments that require a protein preparation whose purity and yield are sufficient for reliable analysis.
The key outcomes are purity, yield, and downstream experimental reliability. Improving expression and solubility can increase the amount available, while refining binding and elution conditions can improve selective recovery. Considering these outcomes together is important because a high amount of recovered protein is not sufficient if unwanted cellular components remain or later experiments become inconsistent.