The RIG-Plasmid:         A Useful Tool To Overexpress
                                              Parasite Genes in Escherichia coli

"RIGging" E. coli for AT-Rich Parasite Gene Overexpression

Background

Overexpression of parasite genes is important for providing sufficient amounts of recombinant protein for biophysical studies, such as X-ray crystallography and NMR, as well as for immunological applications, such as vaccine production and the production of recombinant antigens for the generation of research antibodies. The overexpression of parasite genes in Escherichia coli, in particular those of Plasmodium falciparum, has often been a challenge because of the codon bias of these organisms. P. falciparum has an extremely AT-rich genome of about 80%, which has in many instances made heterologous expression of Plasmodium genes in E. coli very difficult. Certain codons which are preferentially used by P. falciparum are rarely used by E. coli in highly expressed. Rare codons have been shown to greatly diminish expression levels of recombinant protein in E. coli because of translational stalling. One method by which this codon bias has been overcome is to re-engineer the Plasmodium gene to be overexpressed so that it uses the preferred codons of E. coli . This method is both costly and time consuming.

The RIG-plasmid was originally developed to help overexpress P. falciparum genes in E. coli. The RIG-plasmid carries the genes that encode three tRNAs (Arg, Ile, Gly) cloned from E. coli. These genes direct the consitutive expression of tRNAs that recognize the codons AGA/AGG (Arg, R), ATA (Ile, I), and GGA (Gly,G). The increased levels of these three tRNAs may help E. coli better translate parasite mRNAs that are rich in these specific codons, and thus may increase the yields of recombinant parasite proteins.

The RIG-plasmid is compatible with most commercially available vectors and E. coli expression strains. The RIG-plasmid is a simple and convenient tool to test when performing initial overexpression experiments of AT-rich genes. In order to use the RIG-plasmid, all one needs to do is co-transform the RIG-plasmid into E. coli along with the expression vector containing a parasite gene. In certain cases, the time and expense of redesigning parasite genes into the optimal codon bias of E. coli may now be circumvented by use of the RIG-plasmid.

The RIG-plasmid appears to be a useful tool which allows for the overexpression of several AT-rich parasite genes in E. coli. Genes from the following list of parasites oftentimes use the codons AG(A/G), ATA, and GGA. Genes from these parasites overexpressed in E. coli may benefit from use of the RIG-plasmid.

Vector Information

RIG is derived from pACYC184 and carries the argU, ileX, and glyT genes. Since it is a pACYC184 derivative and carries the origin of replication from plasmid p15A, it can co-exist in cells with pBR322-derived vectors which carry the ColE1 origin of replication. (Most overexpression vectors are pBR322-derived vectors.)

RIG carries a chloramphenicol-resistance gene. The plasmid can be maintained in E. coli with chloramphenicol (35-50 ug/mL).

If your parasite gene contains the following codons:
                              AGA (Arginine)
                              AGG (Arginine)
                              AUA (Isoleucine)
                              GGA (Glycine)
then you may want to try using the RIG-plasmid!

     Vector Map:
              For a gif image of the RIG-vector, click here: RIG-Plasmid Map

     Vector Sequence:
              For vector sequence, click here: RIG Nucleotide Sequence

     RIG Protocols:
              For RIG-plasmid protocol, click here: RIG Protocols

RIG-Successes

The RIG-plasmid has been successfully used to overexpress many parasite proteins. Without the RIG-plasmid, these parasite proteins could not be overexpressed. For an example of the power of the RIG-plasmid, click on this image of an SDS-PAGE gel. E-mail us if you have had a success with using the RIG-plasmid! abaca@u.washington.edu. Please reference our work.

RIG-Requests

We are happy to share the RIG-plasmid with researchers in the field of parasitology. Just write a letter in which there is a short description of which genes that will be studied with the RIG-plasmid. Indicate that the RIG-plasmid will be used for research only and not for profit. Also indicate whether your institution is a for-profit or a non-profit institution.

Send the requests to:
                                    Wim G.J. Hol, Professor,
                                    Howard Hughes Medical Institute and Department of Biological Structure
                                    Box 357742
                                    University of Washington
                                    Seattle, WA 98195 USA
                                    FAX (206) 685-7002

References

(1)  A.M. Baca, W.G.J. Hol. "Overcoming Codon Bias: A General Method for High-Level Overexpression of Plasmodium and AT-rich Parasite Genes in Escherichia coli", Abstract # *** American Society for Tropical Medicine & Hygiene 1998 Annual Meeting.

(2)  A.M. Baca, W.G.J. Hol. "Overcoming Codon Bias: A General Method for High-Level Overexpression of Plasmodium and AT-rich Parasite Genes in Escherichia coli", Abstract #1264, American Society for Biochemistry and Molecular Biology 1999 Annual Meeting.

(3)  A.M. Baca, W.G.J. Hol. "Overcoming Codon Bias: A Method for High-Level Overexpression of Plasmodium and AT-rich Parasite Genes in Escherichia coli". International Journal for Parasitology (30), 2000 pp. 113-118,

Click here to download a .pdf version of reference (3).
 

This page was prepared by Arthur M. Baca on December 16, 1999