Recombinant Xanthomonas oryzae pv. oryzae Translation initiation factor IF-2 (infB), partial
Description
Definition and Biological Significance
Recombinant Xanthomonas oryzae pv. oryzae (Xoo) Translation Initiation Factor IF-2 (infB), partial, is a genetically engineered protein derived from the bacterial pathogen Xanthomonas oryzae pv. oryzae, which causes bacterial blight in rice. IF-2 is a critical translation initiation factor in prokaryotes, responsible for binding initiator tRNA to the ribosome and facilitating the assembly of the 70S ribosomal complex during protein synthesis . The recombinant form of this protein lacks the full-length sequence but retains functional domains for biochemical and structural studies.
Production and Purification
The protein is recombinantly expressed in a baculovirus system, which enables post-translational modifications akin to eukaryotic systems. Key production and purification details include:
Table 1: Production and Purification Parameters
| Parameter | Specification |
|---|---|
| Expression System | Baculovirus |
| Expression Region | Amino acids 1–904 (partial sequence) |
| Purity | >85% (verified by SDS-PAGE) |
| Storage | -20°C (short-term); -80°C (long-term) |
| Source Organism | X. oryzae pv. oryzae strain PXO99A |
Purification involves immobilized metal affinity chromatography (IMAC) combined with ion exchange chromatography to achieve high yield (~2.5 mg/L of culture medium) .
Table 2: Functional Assays for Recombinant IF-2
| Assay Type | Methodology | Outcome |
|---|---|---|
| Thermostability | Microscale Thermophoresis (MST) | Retains activity at 25°C in 50 mM NaCl, pH 7.9 |
| Enzymatic Activity | GTPase assay | Pending (no published data) |
Research Applications
This recombinant protein is primarily used for:
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Antibody Production: Serves as an immunogen for generating polyclonal or monoclonal antibodies.
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Mechanistic Studies: Investigating translation initiation in Xanthomonas and related plant pathogens.
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Drug Screening: High-throughput assays to identify inhibitors targeting bacterial translation machinery .
Limitations and Knowledge Gaps
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Partial Sequence: The absence of the full-length protein limits studies on domain interactions.
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Functional Data: No peer-reviewed studies on its enzymatic kinetics or structural biology exist.
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Host Compatibility: Baculovirus expression may introduce eukaryotic post-translational modifications absent in native bacterial IF-2.
Comparative Analysis with Native IF-2
Native IF-2 in X. oryzae pv. oryzae is encoded by the infB gene (locus tag: PXO_RS01025 in strain PXO99A). The recombinant form lacks C-terminal regulatory regions but retains core functional motifs.
Future Directions
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Structural Resolution: Cryo-EM or X-ray crystallography to map tRNA/ribosome-binding sites.
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Pathogenicity Studies: Knockout models to assess IF-2’s role in bacterial virulence and rice infection.
Product Specs
Target Background
KEGG: xop:PXO_01303
