Knowing the sequence of the gene allows the isolation of the cDNA. Unlike the genomic DNA, cDNA's do not contain introns, which are stretches of non-coding sequences interspersed between the exonic (coding) DNA regions. The introns are removed after transcription through a process called splicing. Intron-exon boundaries and splicing mechanisms vary between different species, so that introduction of foreign genomic sequences into a particular species will not automatically result in the synthesis of a functional protein. Introduction and expression of cDNA sequences, however, generally does result in the synthesis of functional proteins. Such proteins are called recombinant proteins.
Recombinant protein can be obtained in large amounts via overexpression of the cDNA of interest in, for example, the bacterium Escherichia coli, the yeasts Saccharomyces cerevisiae and Pichia pastoris, or the Sf9 insect cell line of Spodoptera frugiperda. This is typically achieved by the introduction of a plasmid vector containing the cDNA. He plasmid is a circular DNA replicon that is maintained because it confers a selective advantage, such as an antibiotic resistance. The use of an appropriate promoter results in high expression levels of the cDNA. As a consequence, the abundance of recombinant proteins in these systems is typically much higher than that of the native protein in a crude plant extract. The higher abundance facilitates the purification of the protein to apparent homogeneity. Purification of the recombinant protein can be achieved through traditional biochemical separation techniques, including various types of chromatography (see Section 2). Many of the expression systems, however, are based on so called tags at the N- or C-terminus of the protein. These tags allow binding of the recombinant protein to a specially formulated resin. The crude cell extract containing the recombinant protein is loaded on a column containing the resin, and the recombinant protein becomes bound to the resin, typically via an electrostatic interaction. Undesirable proteins are subsequently removed by washing the column with a buffer that does not disrupt the binding of the recombinant protein. The recombinant protein is eventually eluted with a buffer that disrupts the interaction between the recombinant protein and the resin.
An example of the use of tags to purify recombinant proteins is the addition of six histidine (His) residues to a recombinant protein. The His-residues are encoded by the DNA of the expression vector and will be added to the protein during translation. The purification of the recombinant protein is achieved through immobilized metal affinity chromatography (IMAC). Since His can chelate transition metal ions such as Ni2+, Zn2+, and Cu2+, the His-tag will allow the recombinant protein to bind to a resin containing Ni2+ ions, such as Ni2+-Sepharose. Use of Ni2+ appears to be more successful than the other ions. The recombinant protein is eluted from the column by washing with imidazole, which competes for the Ni2+ binding sites. Alternatively, a low-pH buffer can be used to elute the recombinant protein. The low pH disrupts the electrostatic interactions between the recombinant protein and the Ni2+ on the column, but has the risk of denaturing the protein of interest. In general the presence of the six His-residues has no impact on protein function, but the tag can be removed by endoproteolytic cleavage.
An alternative method to purify recombinant proteins, similar in principle to the His-tag described above, is the fusion with glutathione S-transferase
(GST). This is an enzyme that has high affinity for glutathione. The crude cell extract containing the recombinant GST-fusion protein is loaded on a column of a resin containing glutathione (such as glutathione Sepharose). Undesirable proteins are removed by washing, and the recombinant GST-fusion protein is eluted by washing the resin with reduced gluthathione, which competes with the recombinant protein. The enzyme thrombin is used to specifically remove the GST-tag from the purified recombinant protein. Plasmid vectors to synthesize recombinant proteins with His-tags or GST-fusion proteins, as well as various other types of tags are available from various biochemical supply companies.
Data from in vitro activity assays with these purified recombinant proteins can typically be interpreted much more easily than data obtained from experiments with crude or partially purified protein extracts, because (1) there will be no competing proteins with similar activity present in the assay, and (2) there will no enzymes present that convert the product generated by the enzyme of interest, and hence reduce the effective product concentration. A potential downside of the use of recombinant protein over crude extracts is the fact that critical co-factors that will ensure proper activity may not be present in the purified protein fraction. If that is the case, the researcher will have to empirically determine which co-factor and at what concentration needs to be included in the assay. Another consideration is that the native protein may have undergone post-translational processing, such as acetylation, glycosylation, myristoylation, etc. These modifications may not occur or may not occur properly when the protein is expressed in bacterial, fungal or insect cells. Assuming that these potential problems do not occur or can be dealt with, the availability of pure recombinant protein will enable the determination of substrate specificity, as well as kinetic experiments in which the rate of conversion is measured as a function of time and/or substrate concentration.
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