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Protein purification, gene cloning and sequencing of an acidic endoprotease from Myxococcus xanthus DK101
N Lucas1, C Mazaud-Aujard, L Bremaud
1Institut de Biotechnologie, Faculté des Sciences, Limoges, France.
Abstract:
An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus DK101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe-Met bond of kappa-casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12 kDa and 4.5, respectively. From the N-terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127-bp insert. The nucleotide sequence included an open reading frame (ORF) of 585 nucleotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N-terminal amino acid sequence of the purified MAEP. The polypeptide sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195-amino-acid precursor protein.
Insights
Researchers purified a Myxococcus xanthus acidic endoprotease (MAEP) and identified its gene. This acidic endoprotease enzyme cleaves specific bonds in kappa-casein under acidic conditions.
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- Myxococcus xanthus secretes an acidic endoprotease (MAEP) during vegetative growth.
- Understanding MAEP's function and genetic basis is crucial for microbial studies.
Purpose of the Study:
- To purify and characterize the MAEP from Myxococcus xanthus.
- To isolate and sequence the gene encoding MAEP.
Main Methods:
- Purification using chromatographic procedures.
- Enzyme activity assay on kappa-casein at acidic pH.
- Polymerase chain reaction (PCR) amplification using N-terminal sequence-derived primers.
- Screening of a M. xanthus expression DNA library.
- DNA sequencing and open reading frame (ORF) analysis.
Main Results:
- MAEP was purified to homogeneity, exhibiting specific activity towards the Phe-Met bond of kappa-casein at pH 5.9.
- Estimated molecular mass of 12 kDa and isoelectric point of 4.5.
- A DNA fragment was amplified via PCR and used to isolate a recombinant plasmid containing a 2127-bp insert.
- An ORF of 585 nucleotides was identified, encoding a 195-amino-acid precursor protein with high similarity to the MAEP N-terminus.
- The mature enzyme is predicted to consist of 131 amino acids.
Conclusions:
- The gene encoding MAEP was successfully isolated and sequenced.
- The deduced amino acid sequence provides insights into the MAEP precursor and mature enzyme structure.
- This work provides a foundation for further functional and genetic studies of MAEP in Myxococcus xanthus.