Recombinant Protein Mutagenesis
Recombinant Protein Mutagenesis - Related Videos
09:01
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Cited by 12 •
2011
Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.
11:36
A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
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Cited by 6 •
2016
We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 β-lactamase selected at a clinically relevant dosage of ampicillin are provided.
02:50
Recombinant Protein Production and Recovery from Bacteria-Derived Vesicles
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2025
Source: Streather, B. R., et al. Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli. J. Vis. Exp. (2023)This video demonstrates the production and isolation of recombinant proteins tagged with Vesicle Nucleating Peptide (VNp) from bacterial vesicles. An inducer triggers VNp-tagged protein expression, driving vesicle formation and encapsulation, followed by centrifugation and sonication for efficient protein recovery without cell...
03:39
Generation of an Immunostimulatory Protein-Encoding Recombinant Measles Virus
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2026
Source: Heidbuechel, J. P., Engeland, C. E. Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo. J. Vis. Exp. (2019)This video demonstrates the generation of recombinant measles viruses using liposomes to deliver plasmids encoding the viral antigenome and essential helper proteins into producer cells. The transcribed and replicated RNA leads to the formation of viral particles expressing immunostimulatory and fluorescent reporter proteins. Fluorescence microscopy...
04:51
Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans
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Cited by 6 •
2016
Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.
FAQs
An altered DNA sequence changes the amino acid sequence encoded by the recombinant gene, and that change can modify how the protein folds or performs. Examining binding, catalytic activity, stability, and solubility helps connect a particular sequence change with a measurable property. This sequence-to-function relationship is the central biological value of mutagenesis experiments.
The unmodified recombinant protein provides the reference needed to interpret a mutant’s behavior. If the altered protein shows different binding, catalytic activity, stability, folding, or solubility, the difference can be related to the introduced sequence change rather than treated as an isolated measurement. This paired comparison supports conclusions about which protein properties are sensitive to the altered region.
Expression in bacteria or cultured cells places the modified gene in a biological production system, allowing researchers to obtain the protein for further study. The host is relevant when evaluating production-related outcomes, including folding and solubility, as well as the amount of usable material available for purification and assays. Thus, mutagenesis connects molecular design with protein recovery.
A comparative workflow begins with a planned DNA change, followed by expression of the original and modified genes in a host system. Researchers then purify the resulting proteins and assess relevant functions. Keeping the comparison focused on the altered construct and its unmodified counterpart helps link an observed outcome to the intended molecular change.
Purification prepares the resulting protein for direct evaluation, while functional assays examine properties such as binding or catalytic activity. Additional comparisons can address stability, folding, or solubility, depending on the biological question. Together, these measurements show whether a DNA alteration produced a meaningful protein-level effect rather than merely confirming that a construct was expressed.
In enzyme engineering, researchers can alter selected protein sequences and assess the resulting effects on catalytic activity, stability, folding, or solubility. Comparing modified enzymes with the original helps identify changes associated with improved or altered performance. This makes mutagenesis useful for investigating how enzyme structure relates to function and for developing proteins suited to biotechnology research.
For antibody optimization, sequence changes can be evaluated through properties such as binding, stability, folding, and solubility. In disease-mechanism studies, comparing altered and original proteins can reveal how particular sequence differences affect function. The same experimental logic also supports therapeutic protein research by connecting molecular changes with measurable protein behavior.