subject: Recombinant Protein Expression [print this page] Protein expression is a part of gene expression and helps measure the presence of a particular protein in a specific cell or tissue. To describe a particular gene or protein, its function must be known.
What are recombinant proteins?
In proteomics research, various aspects of the protein are scrutinized. This pertains to the proteins interactions, structure, function, modifications and localizations. To explore the ways in which proteins influence biology, a method of protein production and antibody sequencing is necessary. Since proteins are complex, chemical synthesis is not feasible. Therefore, living cells and their cell biology are leveraged to form and create proteins based on certain genetic blueprints.
On the other hand, DNA is easy to produce synthetically using various techniques and the DNA blueprint of certain genes can be built as templates for protein expression. The protein manufactured from these templates is termed recombinant proteins. This is the conventional method where cells are transfected with a DNA vector with the template followed by cell culture so that the resultant protein can be transcribed and translated. The cells are then lysed to produce the expressed protein which can be purified.
There are many ways to express the cDNA as a recombinant protein and it finds its use in:
Antibody sequencing
Experiments related to protein-protein interaction
Enzyme kinetics
Studying the protein function and structure
Protein crystallization
Recombinant protein expression can relate to two systems: prokaryotic or bacterial and eukaryotic or yeast or mammalian cells.
Prokaryotic protein expression systems
Although there are many benefits in prokaryoticrecombinant protein expression systems such as rapid cell growth, easy to culture and induce expression and purification using a range of kits, the proteins cannot be used for functional and enzymatic studies. This is because they become insoluble in inclusion bodies and cannot be recovered as functional proteins without using complex procedures. This hinders enzymatic studies.
Eukaryotic protein expression systems
Eukaryotic systems for protein expression include yeast, mammalian cells and insect cells which are ideal for recombinant protein expression. The main advantage of the eukaryotic system is the ability to achieve high levels of expression and purification by using special tags. With plasmids that secrete protein into the media, the system can grow without causing cell lysis. Inclusion bodies are non-existent and the proteins are intact and this is critical in the study of protein function and interaction. The only drawback of eukaryotic protein expression is the slow and costly process.
Cell free protein expression systems
Protein expression can also be cell-free, using extracts of the whole cells. Here, the extract has all the necessary components for transcription, translation and further modification. Combined with cofactors, the specific gene blueprint and nucleotides, the extracts are capable of quickly synthesizing the necessary proteins, which helps in.
While this may not be feasible for production on a large scale, cell free protein expression is easy, fast and does not need cell culture.This facilitates the expression of a number of proteins from a variety of recombinant DNA blueprints.
Both prokaryotic and eukaryotic are popular while cell-free protein expression is used only in certain cases.
