PRO Bacterial Expression System

    The PRO Bacterial Expression System is a method by which one can tightly regulate expression of a protein, yet still induce a good yield of that protein from a bacterial plasmid.  This method is unique because it used to be that when one wanted to tightly control the expression of a gene there was always a decrease in the amount of protein for which that gene coded.  Thus, when one tightly regulated the gene one could expect a low yield of its protein, while if one wanted a high yield of it's protein one had to sacrifice control of that gene's expression, which could potentially result in inclusion bodies within the cell ( inclusion bodies are protein packets that accumulate when too much of a particular protein has been made by the cell and the cell cannot use all the protein).  However, with the advent of the PRO Bacterial Expression System it is possible to attain good yields with little protein leakage.  The method is also perfect for the expression of toxic proteins that have the potential to damage the cell, because the PRO system prevents leakage of these proteins.  As an added bonus, this method even allows for the induction of two different proteins at the same time.
    The backbone(s) of the technique are the two plasmid vectors, PROLar and PROTet.  PROTet can induce production of the target protein at a level that is 10% of total protein production.  It is the more inducible of the two vectors.  However, PROLar is more tightly controlled than PROTet, yet still maintains a respectable induction rate of 2% of total protein made.  Thus, PROLar is better suited for production of toxic proteins, while PROTet is better suited for expressing a lot of a non-toxic protein.
     Each vector contains three components that can be removed by restriction enzymes.  Component 1 contains the promoter region, the multiple cloning site, the ribosome binding site, and the Myc tag for protein purification.  Component 2 contains the origin of replication and the transcriptional terminators, T1 and t0.  Component 3 contains the antiobiotic resistance gene.  The multiple cloning site, the ribosome binding site, the Myc tag, and the transcriptional terminators are conserved for both plasmids.  However, the promoters, the ori, and the antibiotic resistance gene are different for the two plasmids.  Finally, each plasmid vector comes in three different reading frames so that the gene sequence being inserted is sure to be in the right reading frame. The following are two cartoons of the plasmid vectors.
(Images courtesy of CLONTECH Labs, Inc.)

    In pPROTet the origin of replication is ColE1 and the promoter is PLtetO-.  ColE1 is a high-copy ori that has a 2,500 fold range of inducibility, meaning one can control protein induction over a wide range by varying the concentration of promoter.  The Pltet0- promoter is a tetracycline-regulated promoter.  This means it only produces protein when it is "turned on" by anhydrotetracycline.  The promoter is further regulated by the Tet repressor, which allows for even greater control of protein induction by turning the gene off (preventing transcription) when it is present.  Thus the highly inducible ColE1 ori and the tightly regulated promoter PLteto- together provide a plasmid that can create many proteins, but only  in the presence of anhydrotetracycline.
    In pPROLar the origin of replication is p15A ori, and the promoter is Plac/ara-1.  The p15A ori is not as inducible as the ColE1 ori in PPROTet, thus its low copy number decreases the chance of protein leakage.  The Plac/ara-1 promoter is based on the lac promoter and is activated by the proteins Arabinose and IPTG.  However, Plac/ara-1 is further controlled by a second  lac operator sequence that is required to allow RNA polymerase to transcribe the nucleotide sequence, thus providing tighter control of protein induction.   For this reason, the pPROLar vector is best suited for the expression of toxic proteins. PPROLar can repress the production of these toxic proteins until the cell has enough materials to quickly produce them in sufficient amounts, then transcription can be turned off so the toxic proteins don't further harm the cell.
    Since the the two plasmids use two different promoters and two different origins of replication one can regulate protein induction independently, allowing for induction of two proteins at the same time in the same DNA strain.  For example, if one uses only anhydrotetracycline then only pPROTet is going to produce protein.  However, if one uses only IPTG/arabinose then the opposite is true, only pPROLar is going to produce protein.  Finally, if one uses both aTc and IPTG/Arabinose then both proteins will be expressed.  The following three figures show protein that is expressed only by pPROTet, only by pPROLar, and both pPROTet and pPROLar at the same time, respectively.

(Images courtesy of CLONTECH Labs, Inc.)
    The Myc tag can be used to isolate your protein by using a monoclonal antibody attached to a bead specific for that protein.  The protein is the only substance in solution that bonds to the beads, so it can be separated from all other cellular machinery that is in the solution.  Then, one can break the hydrogen bonds holding the antibody to the protein, and the protein can be run on a flourograh.  This technique is called immune precipitation.
    Of course, the advances that this method offers are useless if one doesn't know how to insert a nucleotide sequence into a plasmid, or even why one would want to do this.  The PRO Bacterial Expression System is used when one wants to clone a certain nucleotide sequence and express the protein for which this nucleotide sequence codes.  For example, if one wanted to clone the nucleotide sequence for the produtction of luciferase, then one would find this gene sequence and use a restriction enzyme to cut both the bacterial plasmid at the MCS and the nucleotide sequence out of a DNA strand.  One could use either pPROTet or pPROLar, it doesn't matter.  Then, after making sure the gene sequence being inserted is in the appropriate reading frame, the plasmid is mixed with the DNA fragment of interest and the enzyme DNA ligase joins the two strands together.  Since there are many restriction sites for a given restriction enzyme throughout an organism's genome and it is possible for these other sites to bond with the plasmid or even to each other, many combinations of plasmid and gene sequence are possible.  To determine which plasmids bonded to the gene of interest, we transform (term used to describe the ability of bacteria to take up naked DNA) the plasmid into a bacterial cell, in this case E. coli with an antibiotic resistance gene.  Both pPROLar and pPROTet have antibiotic resistance genes, so when placed on a growth medium with an antibiotic, only these two plasmids with immunity to that antibiotic will survive.  Furthermore, one can analyze the restriction sites of the plasmids that survived the antibiotic laden medium in order to confirm the orientation of the insert.  The Clontech procedure also recommends sequencing to confirm the orientation of the insert.  The final step is to induce protein growth by adding the proper concentration of either anhydrotetracycline for pPROTet or arabinose/IPTG for pPROLar, and purify your protein product by using immuno precipitation or SDS-PAGE and Western blotting.  To see a cartoon about cloning into a plasmid, go here.
    The PRO Bacterial Expression system is an exciting and innovative new technique that makes controlling protein induction much easier.


Campbell, N. A.  1996.  Biology, Fourth Edition.  Menlo Park, CA:  Benjamin/Cummings Publishing Company. p. 344-345.

"PRO Bacterial Expression System User Manual."  1998.  CLONTECH web resource-manual.  <> Accessed Feb. 15, 1999.

"Cloning Genes."  1998.  MIT-Web Resource. <>  Accessed Feb. 15, 1999.

Canfield, Elizabeth.  "Sanger Method for DNA Sequencing."  1999.  Lisa Canfield's webpage.  <>  Accessed Feb. 16, 1999.

White, Brian.  "Southerns, Northerns, Westerns, and Cloning:  Molecular Searching Techniques."  1995.  MIT web page. <>  Accessed Feb. 16, 1999.

"SDS-PAGE (Polyacrylamide Gel Electrophoresis)."  1998.  Davidson web page. <>  Accessed Feb. 15, 1999.

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