The Protein Man's Blog | A Discussion of Protein Research

Expression of recombinant proteins with peptide or protein tags is widely used in protein research for three main reasons, ease of purification from a large pool of host proteins, enhancing solubility of the protein and for localization studies. Some important steps to be considered while choosing an expression vector are compatibility of tag sequence with that of the desired protein, codon usage, including linker sequences, peptide cleavage sites and the impact of the tag on the nature of desired protein. Various tags are used ranging from large proteins (Maltose Binding Protein {MBP}) to small peptides (Hexa (6X) Histidine). Tags can be added to either N-terminal side or C-terminal side of the desired protein.

Gene cloning is one of the most important steps in recombinant DNA Technology. Now-a- days researchers are using different cloning techniques depending on purpose, time, cost, ease of use and availability of resources.  A few of the techniques are briefly explained below.

Recombinant proteins are useful for studying biological processes and the structure of protein. Selecting an appropriate expression system is a crucial factor to produce correctly folded protein. Factors to consider when choosing an expression system include:

Biotinylation, also known as biotin tagging/labeling, is the process of attaching biotin to a protein molecule and other macromolecules. Considering its high affinity to avidin and streptavidin (with a dissociation constant of around 10 ^-15 M, biotin-avidin/streptavidin interaction is one of the strongest non-covalent interactions existing in nature), its high specificity and fast on-rate, this technique is perfectly suited for isolating and purifying proteins and protein complexes, as well as identifying protein-protein interactions and post-translational events.