The plasmids for overexpression of proteins , both in form of cDNA clones and clones ORF , have become an indispensable tool in the field of biomedical research, both for analyzing the structure of genes and thus understand its function , as for the study of the biological and dynamic activity of the proteins they encode.
The process of cloning and subsequent sequencing of genes can be avoided by resorting to commercial, ready-to-use plasmids with verified sequences using simple search tools.
Differences Between Cdna Clones And Orf Clones
The fundamental difference is that the ORF (Open Reading Frame) clones contain only the coding sequence of the protein of interest, while the cDNA clones, in addition to the coding sequence, include untranslated sequences from the 5 ‘or 3’ ends (UTRs) or introns.
Let’s analyze each of them in more detail:
1.- Cdna Clones
CDNA clones can be obtained by two different routes: from biological material or by synthetic production. In the first case, the process is quite laborious and consumes many resources, both financial and time consuming. However, the synthetic production of cDNA clones is a process that takes place in three well-defined steps: synthesis, cloning, and validation.
As previously discussed, cDNA clones include the protein coding sequence, as well as the untranslated regions of the 3 ‘(3’UTR) and 5’ (5’UTR) ends, and commercial plasmids can be found for various species. like human, mouse, rat, bovine or zebrafish, among others.
2.- Orf Clones
The ORF clones offer simplification when expressing a protein facilitating a multitude of applications such as detection and / or purification of the protein, intracellular localization, etc.
These ORF clones can be presented in different vectors depending on the expression system that is going to be used to obtain the protein: bacteria, yeasts, mammalian cells, insects, cell-free systems, etc.
Likewise, they can include different tags depending on the application and the advantages sought. Among the most common we can highlight:
- His6 and FLAG : these tags facilitate the purification of the protein by using anti-His6 or anti-FLAG antibodies. Furthermore, due to their small size, the probability that they interfere with the function of the protein is minimal.
- GFP : As it is a fluorescent tag, in addition to facilitating the purification of the protein by means of anti-GFP antibodies, it allows other applications such as the labeling of living cells.
In certain cases, the use of untagged ORF clones can also be chosen , for example, when the tag may interfere with the folding, localization or activity of the protein, or when we have a specific antibody for the localization and protein purification.
At this point, the following question might arise: Which of the two types of plasmids should I choose to carry out my project? Well, as almost always, it depends. It depends on the objective of each experiment. For example, if we simply sought to overexpress the protein under transient conditions, it would suffice to use a cDNA clone . However, in the event that we do not have a good antibody against the protein of interest, we need a simple method to purify it, or we must create a stable cell line for its over-expression, we would opt for a clone. ORF .