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Cloning and expression of the complement fixation antigen-chitinase of Coccidioides immitis
C R Zimmermann1, S M Johnson, G W Martens
1Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616, USA. crzimmermann@ucdavis.edu
Insights
Researchers produced a functional recombinant chitinase from Coccidioides immitis in E. coli. This chitinase serves as a valuable antigen for diagnostic tests and may play a role in fungal development.
Area of Science:
- Mycology
- Molecular Biology
- Immunology
- Biochemistry
Background:
- A chitinase from Coccidioides immitis was previously identified as a key antigen for coccidioidal complement fixation (CF) and immunodiffusion tests.
- Understanding the molecular basis and production of this antigen is crucial for improved diagnostics and understanding fungal biology.
Purpose of the Study:
- To clone, express, and characterize a functional recombinant C. immitis chitinase.
- To confirm the recombinant protein's identity and utility as a diagnostic antigen.
- To investigate the potential role of chitinase in C. immitis spherule development.
Main Methods:
- Degenerate oligonucleotide primers were designed based on the N-terminal sequence of the native chitinase.
- PCR amplification and sequencing were used to obtain a DNA fragment encoding the N-terminus.
- A cDNA library was screened using the PCR product as a probe, leading to the selection and expression of positive clones in E. coli.
Main Results:
- One clone, pCTS 4-2A, expressed an enzymatically active recombinant chitinase that showed identity with the native antigen in immunodiffusion assays.
- The recombinant protein (47 kDa) shared identical characteristics and electrophoretic mobility with the native C. immitis chitinase.
- RNA expression levels correlated with enzyme activity, suggesting chitinase involvement in spherule development.
Conclusions:
- A functional recombinant C. immitis chitinase antigen was successfully produced in E. coli.
- The recombinant chitinase is suitable for serological diagnostic applications.
- The findings suggest a functional role for chitinase in C. immitis spherule-endospore development and maturation.
Abstract:
A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen. In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase. The PCR product was used as a hybridization probe to screen a developing spherule-(lambda)ZAP cDNA library, and three hybridizing clones were selected. These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins. Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen. The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa. When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C. immitis CF-chitinase. Nondenaturing gels of the pCTS 4-2A E. coli lysates and the purified C. immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities. The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae. Thus, a functional recombinant CF-chitinase antigen was produced in E. coli and was used in serological diagnostic applications. These results also suggest a functional role for this chitinase in SE development and maturation.