advances-in-protein-chemistry

purification methodology and laid the foundation for the purification techniques that are in use today. In early 1980s, GE Healthcare
(then Pharmacia) launched a completely integrated chromatography system called FPLC Fast Protein Liquid Chromatography (FPLC)
system. FPLC has since become a useful tool for reproducible chromatographic purification of proteins. During that time, the emphasis
was focused to purifications starting with natural sources, where extremely low concentration of target protein in the source organisms
often made purification of even a few micrograms difficult and time consuming. Nowadays, Process-scale protein purification for large
amounts of proteins used in laundry detergents or for enzymatic synthesis of complicated substances, as well as in biopharmaceuticals,
has become common.
In 1970s, the development of recombinant DNA technology allowed modification and heterologous over expression of a selected
protein. During the 1980s and 1990s affinity tagging of proteins became popular, and it allowed efficient affinity-based purification.
The time of purification was substantially reduced since the same affinity tag could be used on many different proteins. Another
important development in this area was during the Human Genome Project (1990 to 2003) was peptide mass fingerprinting, where
Mass Spectrometry (MS) of peptide fragments of proteins combined with searches of databases with known protein sequences allowed
identification of proteins present in a sample.
At present, protein purification is performed at scales ranging from micrograms and milligrams in research laboratories to tons in
industry. Today, the purification approaches based on affinity tagging of the target protein have revolutionized protein purification.
With its help, many proteins can be purified very easily and efficiently. However, it should be realized that even today, some proteins
may be very challenging to purify in an active and stable form, these include unstable protein complexes, insoluble aggregates, integral
membrane proteins, and proteins that are subjected to multiple post-translational modifications. The challenges in the area of protein
purification that still persist make it not only worthwhile but also essential to gain a deep knowledge about protein purification so that the
available methods can be selected and applied in an optimal way and can be improvised to suit the requirements. In addition, the doors
always remain open for the development of new approaches to protein purification.
Protein Quantification, Yield and Purity
During the protein purification steps, it is always necessary to keep checking:
a. How much fold purification has been achieved till a given point? In other words, one has to look for how much of the contaminating
protein have been removed.
b. What is the yield of the obtained protein? This gives an idea about the percentage of protein that has been retrieved out of the
starting amount or activity.
To achieve these objectives, one needs to have:
a. A method for the estimation of total protein: It is required since the specific assay of target protein gives no information about the
presence of contaminating proteins in the sample.
b. A specific assay method for the desired protein: This is meant for getting an idea of the amount of target protein and degree of
purification achieved.
Protein estimation
There are several approaches to quantify the proteins. However, many of these, for instance, Radiolabelling, Edman degradation,
RP-HPLC, Mass spectrometry, etc., are used less frequently or in special cases due to being more expensive, tedious and complex. In
addition, there are other methods such as Kjeldahl method and Biuret assay that are relatively outdated and therefore, rarely find the use.
In general, spectroscopic and colorimetric methods are most frequently used for estimation of total protein in a sample at a given stage.
Basic principle of some of the commonly used spectroscopic and colorimetric assay methods are briefly mentioned below;
a. Biuret method: Molecules containing two or more peptide bonds form react with copper (II) under alkaline conditions to form a
purple colored complex that absorbs maximally around the wavelength of 540 nm. The intensity of color is directly proportional to the
concentration. The method I rarely used alone due to low sensitivity. However its variants, such as Lowry’s method and Bicinchoninic
acid method (discussed below), are commonly used.
b. Lowry’s method: The method combines the Biuret test (i.e. the reaction of copper (II) with the peptide bonds under alkaline
conditions), with the oxidation of aromatic amino acid residues in the protein. A purple-blue color is obtained by the reduction of
phosphotungstic - phosphomolybdic acid in the Folin–Ciocalteu reagent to heteropolymolybdenum blue by oxidation of aromatic acids,
catalyzed by copper (I), produced in the oxidation of peptide bonds. The absorbance is taken at 660 nm.
c. Bicinchoninic Acid (BCA) method: Like Lowry’s method, BCA assay primarily relies on two reactions. First, the reduction of
cupper (II) to copper (I) by the peptide bond occurs under alkaline conditions. Second, the chelation of each copper (I) ion by two
molecules of BCA, forming a purple-colored product that strongly absorbs light at a wavelength of 562 nm. However, unlike Lowry’s
method, a single reagent containing all the components required for both steps, is sufficient.
d. Bradford’s Dye-Binding method: The method involves the binding of Coomassie Brilliant Blue G-250 to proteins primarily through
ionic interactions with basic amino acid residues (hydrophobic interactions are also believed to exist). The binding of the dye to protein
shifts the absorption maximum of the dye from 465 to 595 nm, which is monitored. The assay is reproducible, single step and rapid (2
minutes incubation). The color remains stable for about one hour.
e. Absorption at 280 nm: Proteins show characteristic absorption at 280 nm. Aromatic amino acid residues Tryptophan, followed by
Tyrosine are major contributors to absorption. Phenylalanine and Disulfide bonds also absorb at this wavelength but very weakly. The
method is simple and does not lead to protein denaturation and sample loss. However, the method is error prone due to the interference
by the contaminants, such as nucleic acids that absorb in similar range.
f. Absorption at 205 nm: Proteins absorb at 205 nm due to the absorption by peptide bonds. It may be useful when there is interference
with absorption at 280 nm or there are very few or no aromatic amino acids in the protein. However, several buffer components and
solvents absorb in this region and may lead to error.
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