A hybrid plasmid for expression of toxic malarial proteins in Escherichia coli
Olivier Cinquin, Richard I. Christopherson and R. Ian Menz*
Department of Biochemistry, The University of Sydney, NSW 2006, Australia
*Corresponding author. Phone: +61 2 9351 2494. Fax: +61 2 9351 4726.
Mol. Biochem. Parasitol. 117(2), pp245-247 (2001)
Abbreviations: IPTG, isopropyl-b-D-thiogalactoside
Key words: over-expression; Plasmodium falciparum; rare codon; toxic protein; pLysS; RIG-plasmid
The human malarial parasite, Plasmodium falciparum, like many parasites, has different codon preferences to those of Escherichia coli . In some cases, codons preferentially used by P. falciparum in highly expressed genes are rare in E. coli . The translation of rare codons can decrease or inhibit expression of recombinant proteins in E. coli . To facilitate over-expression of malarial proteins, Plasmodium genes can be engineered to utilise codons preferred by E. coli. However, site-directed mutagenesis or preparation of synthetic genes is time-consuming and costly. Recently, the codon-bias problem has been solved by introduction of a plasmid encoding extra copies of tRNAs that recognize rare E. coli codons (CodonPlusÔ , Stratagene, USA; RIG plasmid ), allowing over-expression of native Plasmodium genes in E. coli.
We attempted to over-express the malarial enzyme, orotidine-5'-monophosphate decarboxylase (ODCase, EC 220.127.116.11), which catalyses a reaction in de novo pyrimidine biosynthesis . The sequence of this gene from P. falciparum was identified by homology searching genomic data from the International Malarial Genome Sequencing Project (unpublished data). The gene was amplified from P. falciparum strain 3D7 genomic DNA and cloned into the expression plasmid pET-MCSIII  which adds a 6x His tag to the N-terminus of the protein (unpublished data).
The malarial ODCase expression vector (pET-ODC) was used to transform E. coli, BL21 (DE3) carrying the RIG plasmid  (BL21(RIG)), which encodes extra copies of the argU (AGA/AGG), ileX (ATA) and glyT (GGA) tRNA genes. The transformed cells were plated on LB glucose plates containing ampicillin (100 m g/ml) plus chloramphenicol (50 m g/ml), and incubated over-night at 37 °C. The resulting colonies were much smaller than those for BL21(RIG) cells transformed with other malarial expression vectors (unpublished data). These colonies continued to grow extremely slowly after transfer to liquid culture (Fig. 1). The cells reached an optical density (600 nm) of 0.6 after approximately 11 hours and expression was then induced with up to 1 mM IPTG. The cells were harvested after 3 hours induction and no over-expressed protein was detected by SDS-PAGE (not shown). Nickel affinity chromatography of the soluble extract, to concentrate the His-tagged recombinant protein, also failed to detect expression of any malarial ODCase. Similar results were obtained with BL21-CodonPlus™ (DE3)-RIL cells (Stratagene, USA), which encodes extra copies of the argU (AGA/AGG), ileX (ATA) and leuW (CTA) tRNA genes.
The slower growth of E. coli containing pET-ODC compared with cells containing other malarial expression plasmids, suggested that the malarial ODCase was toxic to E. coli and produced at low levels prior to induction with IPTG. Constitutive, low-level, expression of recombinant proteins is a known limitation of the pET expression system (Stratagene, USA), due to leakage of the inducible promoter that controls T7 RNA polymerase . If the recombinant protein is toxic to cells, then low-level expression can be detrimental to growth . Therefore, the pLysS and pLysE plasmids , which constitutively produce low levels of T7 lysozyme, an inhibitor of T7 RNA polymerase , are commonly used for the over-expression of toxic proteins with the pET expression system (Stratagene, USA).
When BL21 (DE3) cells carrying either the pLysS or pLysE plasmids were transformed with pET-ODC, larger colonies and quicker growth rates were observed (not shown). However, no recombinant protein expression was evident after 3 hours induction with 1 mM IPTG. This was attributed to the codon bias of the Plasmodium gene, as a plasmid encoding extra copies of the rare tRNAs was not present in these cells.
It was evident that both the extra copies of the rare tRNAs, and low levels of T7 lysozyme may be required to facilitate expression of the malarial ODCase. However, the pLysS , CodonPlus™ (Stratagene, USA) and RIG  plasmids are all derived from the same progenitor plasmid, pACYC, and therefore, carry the same antibiotic resistance gene (chloramphenicol acetyltransferase) and replication origin, making them incompatible with one another . Hence, we constructed a hybrid plasmid that contained the functionalities of both the pLysS  and the RIG  plasmids.
RIG and pLysS plasmids were propagated in E. coli strain GM48 (ATCC, USA) to prevent methylation, enabling XbaI cleavage, and purified using a GenElute plasmid miniprep kit (Sigma Aldrich, USA). The RIG plasmid was cleaved with XbaI  generating a 1766 bp fragment containing the argU, ileX and glyT genes, which was gel purified using a Qiaquick gel extraction kit (Qiagen, USA). The pLysS plasmid was also restricted with XbaI (unique site) and dephosphorylated with alkaline phosphatase . The purified fragment and the linear pLysS plasmid were ligated overnight at 16 ° C using T4 DNA ligase  and the resulting DNA used to transform E. coli strain DH5a . A positive transformant carrying the pLysS plasmid containing the insert encoding the tRNA genes was identified by restriction mapping using XbaI . The orientation of the insert in this hybrid plasmid (pMICO) is shown in Figure 2 and was determined by restriction mapping with SacI and either ScaI or NcoI .
The insertion point for the tRNA genes into the pLysS plasmid  was selected to maintain the tet and f 3.8 promoters and therefore not alter regulation of the T7 lysozyme gene  (Fig. 2). The tRNA genes remain under the control of their native promoters as in the RIG plasmid .
In contrast to the BL21(RIG) cells transformed with pET-ODC, the BL21(pMICO) cells transformed with pET-ODC yielded large colonies after overnight growth on LB glucose plates containing chloramphenicol (50 m g/ml) and ampicillin (100 m g/ml). Normal exponential growth was observed in liquid culture, with a doubling time of approximately 40 minutes (Fig. 1). Exponential growth of the cells was inhibited by induction of the recombinant protein with 250 m M IPTG, confirming that malarial ODCase was toxic to E. coli. SDS-PAGE analysis of BL21(pMICO) prior to, and 3 hours post induction with 250 mM IPTG showed induction of a protein with a molecular weight of approximately 39 kDa (Fig. 3), corresponding to recombinant malarial ODCase with a predicted molecular weight of 39 810 Da. The induced protein was purified and had full catalytic activity and the appropriate molecular weight, determined by mass spectrometry (not shown).
In summary the new hybrid plasmid, pMICO, facilitates the over-expression of malarial ODCase and is likely to be useful for the over-expression of other genes which utilize rare E. coli codons and whose protein products are toxic to E. coli.
We would like to thank Drs. Alan Cowman and Mark Wickham (Walter and Eliza Hall Institute, Melbourne, Australia) for providing P. falciparum, 3D7 genomic DNA and Prof. Wim Hol and Dr. Arthur Baca (Howard Hughes Medical Institute, University of Washington, Seattle, USA) for providing the RIG plasmid.
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Figure 1. Comparison of growth for BL21(pMICO) and BL21(RIG) cells containing the malarial ODCase expression plasmid. Three single colonies were randomly selected from the pMICO (circles) and RIG (squares) plates and transferred to LB media  containing ampicillin (100 m g/ml) and chloramphenicol (50 m g/ml). The cultures were grown at 37 °C and optical density at 600 nm was measured, the values are means ± standard error for the three cultures. The pMICO data (continuous line) are a good fit (r2 = 0.99) to an exponential curve with a doubling time of approximately 40 minutes. The RIG data (dashed line) are a poorer fit (r2 = 0.92) to an exponential curve with a doubling time of approximately 100 minutes.
Figure 2. pMICO map. The orientation of the genes is shown and the promoters controlling T7 lysozyme, tet and f 3.8, are indicated.
Figure 3. SDS-PAGE analysis of a soluble lysate of E. coli BL21(pMICO) cells transformed with the pET-ODC expression plasmid. Samples were taken from liquid culture (A) prior to and (B) 3 hours post induction and subjected to SDS-PAGE on discontinuous Laemmli gels with a 15% (w/v) resolving gel. Proteins were stained with Coomassie brilliant blue. The migration positions and molecular weights of standard proteins are indicated and the over-expressed malarial ODCase is marked with an arrow.