During the study of horizontal gene transfer of the aro A CP4 gene encoding CP4 EPSPS from genetically modified feed through gastrointestinal tract to bacteria living in animal gut we have observed beside full length gene also functional truncated one present in bacteria [1]. The codon usage of both forms was originally optimized for plant [2]. Therefore we have used two different E. coli expression systems (with tac promoter and T7 promoter) to compare enzymatic properties and functional activities of both forms of CP4 EPSPS protein.
Results
For the protein expression we have prepared pKK1 and pKK2 plasmids – tac expression vectors derived from pKK233-2 plasmid, and pMD2 and pMD72, T7 expression vectors derived from pET28a plasmid. Gene aro A CP4 (full length as well as truncated one) from its ATG codon was amplified by PCR from GM plant and cloned into NcoI and HindIII sites of both expression vectors (see Figure 1). We have compared phenotypic properties of full length and truncated CP4 EPSPS forms by growth on solid M9 medium. The results from this experiment are summarized in Table 1. We have also investigated growth curves of E. coli Δaro A strains containing either pKK1 or pKK2 plasmid on liquid M9 medium without aromatic compounds. Received results are shown on Figure 2. We have received suitable expression levels of both proteins with T7 expression system for further partial protein purification based on Ni2+ His tag procedure and comparative enzymatic assay.
Figure 1
Physical maps of plasmids pKK1, pKK2, pMD2, pMD72.
Table 1
Complementation test of the CP4 aro A phenotypes.
Plasmids in E. coli strain
2% Glp
3% Glp
4% Glp
MM
pKK1 in SV1TcR
+++
++
+
+++
pKK2 in SV1TcR
+++
++
+
+++
pKK233-2 in SV1TcR
-
-
-
-
pKK233-2 in JM106
ND
ND
ND
+++
pKK1 – pKK233-2 with full length cp4epsps, pKK2 – pKK233-2 with truncated aroA CP4, Glp – glyphosate, + indicates the intensity of the growth, – indicates the absences of growth, MM – minimal M9 medium without aromatic amino acid supplements, ND – not done. SV1TcR strain carries Δaro A mutation.
Figure 2
Growth curves of E. coli SV1TcR with plasmids pKK1 and/or pKK2 in M9 liquid medium. Both strains were grown in concentration of 0.5% glyphosate.
Conclusion
We can conclude from our results that truncated form of CP4 EPSPS (shorter to 31 amino acid residues on N-terminal end) can confer full function of native protein and these findings also should be taken into account in risk assessment of possible HGT from GM food/feed. The enzymatic properties and comparison of both EPSPS forms will be subject of further studies.
Declarations
Acknowledgements
This work was supported by grant of Slovak Grant Agency APVT-20-17102.
Authors’ Affiliations
(1)
Department of Molecular Biology, Comenius University
References
Stuchlík S, Natarajan S, Turña J: Spontaneously generated truncated forms of CP4 EPSPS protein. FEBS Journal. 2005, 272: Supplement 1. Abstracts 30th FEBS Congress and 9th IUBMB Conference, Budapest, Hungary 2–7 July 2005Google Scholar
Harrison LA, Bailey MR, Naylor MW, Ream JE, Hammond BG, Nida DL, Burnette BL, Nickson TE, Mitsky TA, Taylor ML, Fuchs RL, Padgette SR: The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice. J Nutr. 1996, 126: 728-740.Google Scholar
