Protein Characterization and Purification Methods
Protein characterization involves the use of experimental methods that allow for the detection and isolation of a protein and its purification, as well as the characterization of its structure and function. The success of newer advanced, sensitive methods and techniques was the result of recent advancements made in biochemistry, biotechnology, molecular biology, molecular medicine and other related sciences. These were specially developed to allow the purification of amino acids, peptides, proteins, and metabolites. The scope of methods used for protein purification can vary widely ranging from simple one-step precipitation procedures to large-scale validated production processes. To reach the desired purity more than one step is often necessary. The key to a successful purification is the selection of the most appropriate techniques, to optimize and combine them in a logical way to minimize required purification steps and to maximize yields. Most purification approaches involve some form of chromatography. Therefore, chromatography has become an essential tool for protein purification. The combination of different chromatography techniques with different selectivity allows for the design of powerful purification schemes to purify any biomolecule.
The development of recombinant DNA techniques revolutionized the production of proteins in large quantities. Often recombinant proteins are produced such this facilitates their subsequent chromatographic purification. This approach has made purification simpler but has not removed all challenges. Often contaminants are still present, and problems related to solubility, structural integrity, and biological activity can still exist. However, in many purification schemes, a protein maybe is first purified to a homogeneous product.
The analysis of the resulting protein products can be complex and challenging. However, accurate characterization of the final protein that was either purified from natural sources or expressed in different cell cultures or as a part of a drug development process is an essential step. The complex structure and larger size of proteins, as well as the intrinsic nature of each protein, makes the characterization of proteins inherently more complicated than the characterization of smaller molecules. Furthermore, micro heterogeneities are commonly observed even in highly purified protein fractions. Process- and product-related impurities or contaminants can make the purification process difficult. Monitoring the final protein product for all potential contaminations is mandatory. Each new purification protocol requires the implementation of the appropriate specifications. Sometimes, to ensure reproducibility, even a simple analytical assay, like quantification, can become extremely complex. During protein expression, purification and functional in-vitro and in-vivo assays many analytical questions and quality control issues will need to be addressed.
In general, a protein purification protocol involves the isolation of proteins from their source, either from plants, animals, bacteria, viruses, and other sources. For example, serum albumins, antibodies, and other proteins can be purified from serum, ascites fluid, culture supernatant of a cell line, and others. To use the purification of antibodies as an example, purification protocols can cover a wide range. To achieve a final product or formulation that fits the desired purity criteria, very crude to highly specific preparations will be needed. For example, a crude preparation protocol may include the precipitation of a subset of total serum proteins that may include the presence of immunoglobulins as well. However, a more general protocol may include the use of an affinity purification step to purify the desired antibody classes selectively, for example, IgGs. For a more specific approach, the knowledge of the antigen specificity will allow to tailor the affinity purification step needed to only purification of only those antibodies in a sample that bind to a particular antigen molecule. However, the desired purity level depends upon the intended application(s).
The availability of powerful computers allows now researchers to use computational methods for the analysis of proteins in-silico starting with the known protein sequence. Recent progress made in the field of genomics made the sequences of whole genomes available that now allows in-silico analysis of gene products. Selected sequences derived from nucleic acid information with the help of bioinformatics software can be sequences of known or theoretical proteins. Furthermore, the increase in computation power enable computational analysis of many modern experimental methods to speed up the interpretation of the raw experimental data.
Biochemistry studies the structure, composition, and chemical reactions of substances in living systems the majority of which contain proteins. The sciences of molecular biology, immunochemistry, neurochemistry, bioinorganic, bioorganic, and biophysical chemistry are all included in biochemistry. The term “biochemistry” appears to have been first used in 1882. The notion "biochemistry" or the word "biochemistry" was first proposed in 1903 by the German chemist Carl Neuberg. However, starting in the mid-20th century many new techniques have been developed. These include techniques such as chromatography, electron microscopy, NMR spectroscopy, radioisotopic labelling, protein identification and sequencing, UV spectroscopy, X-ray diffraction, and molecular dynamics simulations and many more. Protein sequencing is now possible with the help of the classical Edman chemistry or the more recently developed technique called "liquid chromatography tandem mass spectrometry (LC-MS/MS)". These methods were used to discover and analyze in more detail many molecules and metabolic pathways of the cell including glycolysis and the Krebs cycle (citric acid cycle).
The metabolic health of the proteome directly influences the health of the cell and the lifespan of an organism. Multiple and diverse challenges during the life of an organism can perturb the homeostasis of the proteome. The term "proteome" was first coined by Marc Wilkins in 1994 during a symposium on "2D Electrophoresis: from protein maps to genomes" held in Siena in Italy. He and others published a paper in 1995 that included parts of Wilkins's Ph.D. thesis. In this publication, a protein map of the smallest known self-replicating organism, Mycoplama genitalium (Class: Mollicutes) was described, revealing a high content of acidic proteins. The term, proteome, was used to describe the entire complement of proteins expressed by a genome, cell, tissue or organism. Therefore, the science of proteomics aims to determine and characterize the set of proteins expressed by the genetic material present in an organism under a set of environmental conditions. In other words, proteomics aims to study the complete protein complement expressed by the genome of an organism at a given time, now also called the complete proteome. Therefore, classical protein analysis has evolved now to modern proteomic analysis or “proteomics”.
A complete characterization of a given protein may involve the use of multiple techniques. A thorough characterization of the studied protein can be achieved by the quantitative determination of the protein concentration, for example by using amino acid analysis. Or, the measuring of the absorbance of the protein in solution at 280 nm using an ultraviolet (UV) spectrophotometer. The intact molecular weight can be measured using mass spectrometry. Next, the protein sequence can be elucidated using N-terminal Edman chemistry based protein sequencing, and N- and C-terminal sequencing by mass spectrometry. Additional techniques for further characterizations are peptide mapping using chemical or enzymatic digest in combination with reversed phase liquid chromatography and tandem mass spectrometry. For this, often the protein has first to be isolated or separated from other proteins, metabolites or impurities present in the source. Routinely techniques such as polyacrylamide based gel electrophoresis (1D or 2D PAGE) in combination with blotting techniques (Electro-blotting or Western blotting) are used. Alternatively, liquid chromatographic methods (1D or 2D, ion exchange, reversed phase, etc.) may offer a more detailed analysis.
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