Special Feature

User Panel

My Panel

My Panel

Bookmark Science Articles

Recent News
Bookmark / Share This Science Site

Recombinant Protein Expression

Copyright © Molecular Station 2012

When you want to characterise a gene or protein of interest, you must first study its function. In this molecular era, obtaining a cDNA of your gene of interest is not difficult.

To express the cDNA as a protein, ie. a recombinant protein, one can then easily perform functional studies using the recombinant purified protein.

Once you have a purified protein you can conduct:

  • Protein-protein Interaction Experiments
  • Enzyme Kinetics
  • Functional Studies of the Protein
  • Structural Studies - including protein crystallization, protein structure and NMR.
  • You can also use the protein to make antibodies for further experiments.

 

There are two main systems for the expression of recombinant protein. Once you get your cDNA cloned, you must decide where you want to amplify your protein. This will be either a prokaryotic (bacterial) or eukaryotic (usually yeast or mammalian cell) system. The choice of your system will decide which vector you will need to clone your cDNA into as there are different promoters which function in E.Coli and others that work best with yeast or mammalian systems.

So which protein expression system will you use?

 Systems for Protein Expression

Prokaryotic Protein Expression Systems

Prokaryotic recombinant protein expression systems have several advantages. These include ease of culture, and very rapid cell growth meaning you won't have to wait long to get protein from bacterial systems once you clone your cDNA. Expression can be induced easily in bacterial protein expression systems using IPTG. Also, purification is quite simple in prokaryotic expression systems and there are a plethora of commercial kits available for recombinant protein expression.

On the other hand, if you need to use your proteins for functional or enzymatic studies prokaryotic systems are a problem as most proteins become insoluble in inclusion bodies and are very difficult to recover as functional proteins. Furthermore, most if not all post-translational modifications are not added by bacteria and therefore your protein of interest may not be functional. Enzymatic studies thus may be unfruitful.

Eukaryotic Expression Systems

Eukaryotic genes are not really “at home” in prokaryotic cells, even when they are expressed under the control of the prokaryotic vectors.  One reason is that E. coli cells frequently recognize the protein products of cloned eukaryotic genes as outsiders and destroy them.  Another is that prokaryotes do not carry out the same kinds of posttranslational modification as eukaryotes do.  For example, a protein that would ordinarily be coupled to sugars in a eukaryotic cell will be expressed as a bare protein when cloned in bacteria.  This can effect a protein’s activity or stability, or at least its response to antibodies.  A more serious problem is that the interior of a bacterial cell is not as conducive to proper folding of eukaryotic proteins as the interior of a eukaryotic cell.  Frequently, the result is improperly folded, inactive products of cloned genes. 

Eukaryotic systems for the expression of protein include:

  • yeast
  • mammalian cells
  • baculovirus cells (insect)

All these systems are great eukaryotic systems for the expression of recombinant proteins.
Advantages of eukaryotic protein expression systems include the fact that you can get very high levels of expression. The proteins are easy to purify using special tags which are included into the vectors including His, Myc and other tags.

You can even purchase plasmids which secrete your protein into the media. Therefore you can keep growing your system and collecting the media without lysing your cells. There are no inclusion bodies to worry about and your proteins have intact post-translational modifications. These are vital if you are studying the function of a protein and/or protein-protein interactions.

The disadvantages of eukaryotic protein expression systems include the fact that eukaryotic cells do grow slower than prokaryotic cells.

Expression Vectors with Strong Promoters

The main function of an expression vector is to yield the product of a gene- usually, the more product the better.  Therefore, expression vectors are ordinarily equipped with very strong promoters; the rationale is that the more mRNA that is produced, the more protein product will be made.
One such strong promoter is the trp (tryptophan operon) promoter.  It forms the basis for several expression vectors, including ptrpL1.  It has a trp promoter/operator region, followed by a ribosome binding site, and can be used directly as an expression vector by inserting a foreign gene into the ClaI site. Alternatively, the trp control region can be made “portable” by cutting it out with ClaI and HindIII and inserting it in front of a gene to be expressed in another vector

 

Inducible Expression Vectors

It is usually advantageous to keep a cloned gene represed until we are ready to express it.  One reason is that eukaryotic proteins produced in large quantities in bacteria can be toxic.  Even if these proteins are not actually toxic, they can build up to auch great levels that they interfere with bacterial growth.  In either case, if the cloned gene were allowed to remain turned on constantly, the bacteria bearing the gene would never grow to a great enough concentration to produce meaningful quantities of protein product.  The solution is to keep the cloned gene turned off by placing it downstream of an inducible promoter that can be turned off.
The lac promoter is inducible to a certain extent, presumably remaining off until stimulated by the synthetic inducer isopropylthiogalactoside (IPTG).  However, the repression caused by the lac repressor is incomplete, and some expression of the cloned gene will be observed even in the absence of inducer.  One way around this problem is to express our gene in a plasmid or phagemid that carries its own lacI gene, as pBS does.  The excess repressor produced by such a vector keeps our cloned gene turned off until we are ready to induce it with IPTG.
Another strategy is to use a tightly controlled promoter as the λ phage promoter PL. Expression vectors with this promoter/operator system are cloned into host cells bearing a temperature-sensitive λ repressor gene (c1857).  As long as we keep the temperature of these cells relatively low (32°C ), the repressor functions, and no expression takes place.  However, when we raise the temperature to the nonpermissive level (42ºC), the temperature-sensitive repressor can no longer function and the cloned gene is induced.

 

Expression Vectors that Produce Fusion Proteins

When most expression vectors operate, they produce fusion proteins.  This might at first seem a disadvantage because the natural product of the inserted gene is not made.  However, the extra amino acids on the fusion protein can be a great help in purifying the protein product.


Consider the oligo-histidine expression vectors, one of which has the trade name pTrcHis.  These have a short sequence just upstream of the multiple cloning site that encodes a stretch of six histidines.  Thus, a protein expressed in such a vector will be a fusion protein with six histidines at its amino end.  Why would we want to attach six histidines to our protein?  Oligo-histidine regions like this have a high affinity for metals like nickel, so we can purify proteins that have such regions using nickel affinity chromatography.  The beauty of this method is its simplicity and speed.  After the bacteria have made the fusion protein, we simply lyse them, add the srude bacterial extract to a nickel affinity column, wash out all unbound proteins, then release the fusion protein with histidine or a histidine analog called imidazole.  This procedure allows us to harvest essentially pure fusion in only one step.  This is possible because very few if any natural proteins have oligo-histidine regions, so our fusion protein is essentially the only one that binds to the column.


What if we want our protein free of the oligo-histidine tag? 

The designers of these vectors have thoughtfully provided a way to remove it.  Just before the multiple cloning site, there is a coding region for a stretch of amino acids recognized by the proteolytic enzyme enterokinase.  So we can use enterokinase to cleave the fusion protein into two parts:  the oligo-histidine tag and the protein we want. The site recognized by enterokinase is very rare, and the chance that it exists in our protein is insignificant.  Thus, our protein should not be chopped up as we are removing its oligo-histidine tag.  If we want, we can run the enterokinase-cleaved protein through the nickel column once more to separate the oligo-hisitidine fragments from the protein of interest.


λ phages have also served as the basis for expression vectors;  one designed specifically for this purpose is λgt11.  This phage contains a lac control region followed by the lacZ gene.  The cloning sites are located within the lacZ gene, so products of a gene inserted into this vector will be fusion proteins with a leader of B-galactosidase.
The expression vector λgt11 has become a popular vehicle for making and screening cDNA libraries.  λgt11 allows us to screen a group of clones directly for the expression of the right protein.  The main ingredients required for this procedure are cDNA library in λgt11 and an antiserum directed against the protein of interest.
We plate our λ phages with various cDNA inserts and blot the proteins released by each clone onto a support such as nitrocellulose.  Once we have transferred the proteins from each plaque to nitrocellulose, we probe with our antiserum.  Next, we look for antibody bound to protein from a particular plaque, using labeled protein A from Staphylococcus aureus.  This protein binds tightly to antibody and labels the corresponding spot on the nitrocellulose.  We detect this label by autoradiography or by phosphorimaging, then go to our master plate and pick the corresponding plaque.  Note that we are detecting a fusion protein, not the protein of interest itself.  Furthermore, it does not matter if we have cloned a whole cDNA or not.  Our antiserum is a mixture of antibodies that will react with several different parts of our protein, so even a partial gene will do, as long as its coding region is cloned in the same orientation and reading frame as the B-galactosidase coding region.

For a quick recombinant protein expression review see:

Recombinant Protein Systems

Copyright © Molecular Station 2012

 

Science News

For science news click here:Science News