Cloning And Expression Of A Subtilisin Like Serine Protease Gene From Chaetomium Globosum In Escherichia Coli

Chaetomium globosum parasitizes a large variety of phytopathogenic fungi by producing and releasing different hydrolytic enzymes and antibiotics which inhibit fungal growth. A number of studies have shown that the enzymatic activities in C. globosum enable the fungi to penetrate a suitable host and use the host's biomass as a source of nutrients. Due to such mycoparasitic activity, C. globosum has been used for the biological control of a large variety of phytopathogenic fungi. Documented evidence exists on the ability of C. globosum to reduce pathogen inoculum and disease incidence of a number of agronomically important pathogens. Certain biocontrol related genes have been cloned from the fungus; for instance comprehensive work has been done on the cloning of chitinases and xylanases from C. globosum. However, not much research has been done on proteases secreted by Chaetomium strains, despite their significant role in biological control.The DNA and cDNA sequences of a subtilisin-like serine protease gene, ser, were constructed from C. globosum. The 5'- and 3'-terminal regions of the full-length cDNA were successfully cloned from the C. globosum EST collection by combining bioinformatical analysis and molecular biology techniques. The DNA and cDNA sequences of ser gene were then amplified from C. globosum using PCR based on the sequences of the two ESTs. The nucleotide sequences of the cDNA and mRNA were submitted to the NCBI database under the accession numbers FJ589720 and FJ589721, respectively. The DNA and cDNA sequences of the ser gene are 1682-bp and 1608-bp in length, respectively, and encode 535 amino acid residues with a molecular weight of 57.2 kDa and isoelectric point of 5.93. The DNA sequence of the ser gene is composed of 2 exons and 1 intron. The two exons are 372 and 1236-bp in length, respectively, whereas the intron is 74-bp in length. The splice sites between the exons and introns follow the GT and AG splicing rule. SignalP analysis predicted a 16 amino-acid N-terminal signal sequence and a signal sequence cleavage site located between two Ala amino-acids at positions 15 and 16 demonstrating that the protein is a secreted enzyme. The deduced amino acid sequence of the ser gene showed more than 80% similarity with a majority of serine protease sequences from other fungi and it belongs to the Peptidase S8 superfamily.A recombinant plasmid was constructed using the vector pET-52b(+) under the control of a strong bacteriophage T7 transcription promoter and it was cloned by transforming into Escherichia coli Top10 strain. The recombinant plasmid pET-52b/Ser from positive transformants was then transformed into the expression vector E. coli BL21 for protease expression. For both cloning vectors, positive clones were verified by colony PCR and restriction enzyme double digestion. The transcription and expression of ser was verified by SDS-PAGE, the results showing however, that the target gene could not be clearly viewed on the SDS gels. A study of the induced cultures showed that the enzymatic activity of ser gene transformants was limited with the highest level attained being 0.171U/10ml after 5h of culture and in accordance with the expression of mRNA. The optimum pH for the concentrated enzyme was pH 4.5 and the optimum temperature was 40°C.The present study has demonstrated that at temperatures above 60°C, the enzyme lost its activity rapidly. The protease was stable in a pH range of 3.0–6.0 and the enzyme retained 95% of the full activity in the reaction buffer at pH 4.6.Although the attempt to express the ser gene at high levels in E. coli have not been entirely successful, further optimization is possible. The successful cloning and expression of the gene from C. globosum provides a basis for the study and exploitation of proteases in biological control.