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Single-Molecule Analysis by Biological Nanopores
Published in Shuo Huang, Single-Molecule Tools for Bioanalysis, 2022
An alternative means of preparing biological nanopores is by prokaryotic expression followed with purification by fast protein liquid chromatography. In this approach, a tag protein such as His-tag [54] or Strep-tag [36] is normally placed on either terminus of the target protein for later purification purposes. The prokaryotic protein expression system can efficiently produce several mg of protein in 3–4 days. However, if the target protein is cytotoxic to the host cell during expression, difficulties may ensue. This method also requires more complicated purification procedures to eliminate interferences from the background proteins generated by the host cell. Site-directed mutagenesis is sometimes needed to optimize the performance of the protein nanopore [55]. This can be accomplished by direct synthesis of the mutated gene or with a site-directed mutagenesis kit.
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
The development of affinity tags has led to general methods for the rapid enrichment and purification of heterologously expressed proteins. The most common of these tags is the His-tag. In His-tag protein purification, the protein is bound to a column displaying either immobilized nickel (II) ions or cobalt (II) ions and subsequently competed off with high concentrations of imidazole (Figure 5.8). A wide variety of metal chelate resins are available for protein purification, ranging from low-pressure/low-flow-rate resins to high-pressure/high-flow-rate resins. For many applications, a single affinity purification step employing an affinity tag produces protein that is sufficiently pure for subsequent analysis.
Proteins and Proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
To perform in vitro analysis, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell’s membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins, membrane lipids and proteins, cellular organelles, and nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge, and binding affinity. The level of purification can be monitored using various types of gel electrophoresis (Figure 3.13) if the desired protein’s molecular weight and isoelectric point are known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Additionally, proteins can be isolated according their charge using electrofocusing. For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a “tag” consisting of a specific amino acid sequence, often a series of histidine residues (a “His-tag”), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded. Several different tags have been developed to help researchers purify specific proteins from complex mixtures.
Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application
Published in Preparative Biochemistry & Biotechnology, 2023
Kashif Maseh, Syed Farhat Ali, Shazeel Ahmad, Naeem Rashid
Optimization of expression conditions can have several benefits e.g., equipment can be free for further use along with reduction in the cost of production by reducing the electricity cost as well as long term life of the equipment. The contaminant level and the cost associated with downstream processing is also reduced. In case of Pca-Pol, expression optimization and affinity purification can save 87% of the cost when compared to untagged enzyme. Addition of His-Tag is a useful mechanism for affinity purification of recombinant proteins.[22] After optimizing gene expression, we purified His-tagged Pca-Pol by IMAC. 92% of total enzyme activity was recovered (9000 U per liter) in the single-step purified fraction with a 7.4 fold purification. Whereas for untagged Pca-Pol, the recovery of the purified enzyme was 60% (nearly 6150 U per liter of the culture).[30] Hence expression optimization and affinity purification improved the yield and recovery of Pca-Pol as compared its untagged version. In this current study, we recovered 9000 U of purified Pca-Pol per liter of the culture. 2 U of Pca-Pol were used for a PCR mixture of 20 µL which produced comparable results as those of Pfu and Taq DNA polymerases. So, even if a larger volume of PCR reaction mixture is to be used, this preparation of Pca-Pol (from one liter of the culture) will be sufficient for more than 1500 PCR reactions.
Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii
Published in Preparative Biochemistry and Biotechnology, 2018
M. Mervan Çakar, Juan Mangas-Sanchez, William R. Birmingham, Nicholas J. Turner, Barış Binay
The C-terminal His-tag allowed purification of the protein using immobilized metal affinity chromatography (Figure 1c). This construct has a calculated molecular weight of 79,701 Da, including the C-terminal hexa histidine-tag. As determined by SDS-PAGE analysis, the purified protein had a molecular mass of about 80 kDa, which is close to the predicted molecular mass. The purity and quantity of ClFDH from this expression were sufficient to allow further characterization (Figure 1c, lanes E4–E6).