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Using protein as starting material

There are several things worth mentioning if our starting material will be the protein itself.

First of all, we might not know the sequence of the protein. If this is the case, we can usually find it out by searching through online protein databases. The most popular database containing the biological information is National Center for Biotechnology Information (NCBI). We can find out the sequence of our protein simply by searching for it in the existing database.

The sequence we will get will be composed out of letters representing amino acids. However, in order to successfully clone the gene for expression, we will need the DNA sequence. We can get it from the amino acid sequence by using one of the available online software for reverse translation (like the software Reverse Translate on Expasy.org).

The thing is that we will not get 100% “certain” DNA sequence since some amino acids have more codons. For example, Serine can be coded from the bases AGT and AGC. What we will get instead is the probability that the amino acid from the protein of our interest will be coded by a certain codon. This probability, in the software Reverse Translate, is based on the Codon Usage Database. More precisely, it is based on all of the E. coli sequences present in GenBank. E. coli is used since it is a model organism and its database of sequences is pretty large, so the results obtained by the Reverse Translate software are realistic.

We will usually be able to find the sequence of our protein in the existing databases. However, if we do not, we can find it out by sequencing our protein. Two most popular protein sequencing methods are Edman Degradation and Mass Spectrometry. Once we have the sequence, we can reverse translate it to get the DNA sequence and successfully use it in the gene cloning, or basically any other experiments we might be doing.