Molecular cloning and expression of recombinant Trichoderma harzianum chitinase in Pichia pastoris

Muhammad Saleem Khan, Anwar Khan, Olawale Samuel Adeyinka, Iqra Yousaf, Saman Riaz, Bisma Bashir, Muhammad Tariq, Bushra Tabassum


Background: The importance of chitinases over the years had attracted huge biotechnological attention because its usage cut across wide range of field. It plays a significant role in the defensive mechanism against fungal pathogens.

Methods: In this study, an endochitinase gene was isolated from Trichoderma harzianum, and characterized in-silico by using various bioinformatics tools. Further, the gene was cloned in eukaryotic expression vector (pPICZA) under the control of AOX1 promoter for recombinant expression in Pichia pastoris GS115 host strain.

Results: The chitinase cDNA was ~1000 bp long, while in-silico studies revealed an open reading frame of 888 bp encoding 295 amino acids with a calculated molecular mass of 37332.76 Da and an estimated isoelectric point of 4.07. Recombinant chitinase protein expressed intracellularly and revealed high expression in P. pastoris host. The 37 kDa recombinant chitinase protein developed with antigen antibody confirmed its expression in P. pastoris.

Conclusion: Conclusively, T. harzianum derived chitinase gene was successfully over expressed in P. pastoris where recombinant protein was expressed intracellular in the form of inclusion bodies.

Keywords: Trichoderma harzianum; Chitinase; Pichia pastoris

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