www.als-journal.com/ ISSN 2310-5380/ May 2020 Full Length Research Article Advancements in Life Sciences – International Quarterly Journal of Biological Sciences ARTICLE INFO Open Access Date Received: 22/02/2019; Date Revised: 21/04/2020; Molecular cloning and expression of recombinant Trichoderma Date Published Online: 25/05/2020; harzianum chitinase in Pichia pastoris Authors’ Affiliation: 1 2 1 1 1 1. Centre of Muhammad Saleem Iqbal Khan , Anwar Khan , Olawale Samuel Adeyinka , Iqra Yousaf , Saman Riaz , Excellence in Molecular 1 1 1 Biology (CEMB), University Bisma Bashir , Muhammad Tariq , Bushra Tabassum* of the Punjab, Lahore - Pakistan 2. Department of Abstract Microbiology, BUITEMS, Quetta - Pakistan ackground: 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 *Corresponding Author: Bushra Tabassum against fungal pathogens. Email: B [email protected] Methods: In this study, an endochitinase gene was isolated from Trichoderma harzianum, and characterized in- How to Cite: silico by using various bioinformatics tools. Further, the gene was cloned in eukaryotic expression vector Khan MSI, Khan A, OS (pPICZA) under the control of AOX1 promoter for recombinant expression in Pichia pastoris GS115 host strain. Adeyinka, Yousaf I, Riaz S, Bashir B, Tariq M, Tabassum B (2020). Risk factors Results: The chitinase cDNA was ~1000 bp long, while in-silico studies revealed an open reading frame of 888 associated with relapse of drug dependence after bp encoding 295 amino acids with a calculated molecular mass of 37332.76 Da and an estimated isoelectric treatment and rehabilitation in areas under the influence point of 4.07. Recombinant chitinase protein expressed intracellularly and revealed high expression in P. pastoris of war on terror. Adv. Life host. The 37 kDa recombinant chitinase protein developed with antigen antibody confirmed its expression in P. Sci. 7(3): 122-128. pastoris. Keywords: Trichoderma harzianum; Conclusion: Conclusively, T. harzianum derived chitinase gene was successfully over expressed in P. pastoris Chitinase; Pichia pastoris where recombinant protein was expressed intracellular in the form of inclusion bodies. als Advancements in Life Sciences | www.als-journal.com | May 2020 | Volume 7 | Issue 3 122 You’re reading Molecular cloning and expression of recombinant Trichoderma harzianum chitinase in Pichia pastoris amplified by successive PCR and efficiently expressed Introduction in P. pastoris GS115. The expression of chitinase Chitin is a linear polymer of N-acetyl-β-d-glucosamine protein in a heterologous system permits future (GlcNAc) units linked with β-1-4 glycosidic bond and functional enhancement in biotechnological application encompass enormous glycans families [1]. It is a main against phytogenic fungi. constituent of cell wall in many microbes and provides strength to the exoskeletons of crustaceans, insects, and fungus cell walls. Scientists over the years have utilized Methods the advantages of chitin hydrolyzing enzymes Fungal strain and culture conditions (endochitinases, exochitinases and N- T. harzianum chitinase producing mutant was obtained acetylglucosaminases) to produce many kinds of its from the Institute of Agricultural Sciences, University of derivatives with potential applications in medicine, food, the Punjab, Pakistan. Potato dextrose agar (PDA) cosmetics, biocontrol, health products and medium was used to maintain and propagate fungal environmental protection [2, 3]. Different enzymes cultures. The expression vector pPICZA and yeast cleave chitin in different manners: Endochitinases expression host Pichia pastoris GS115 were obtained randomly digest the chitin chain from β-1,4-glycosidic from Pichia EasySelect Kit (Invitrogen). bonds while exochitinases digest from the nonreducing Trichoderma harzianum chitinase amplification and end of chitin chain and diacetyl-chitobiose (GlcNAc2) is cloning in pCR2.1 vector formed; which is converted into GlcNAc by T. harzianum hyphae were homogenized in liquid acetylglucosaminases or from the nonreducing end of N- nitrogen with pestle mortar. TRI Reagent (Sigma- acetyl-chito-oligosaccharides [4]. Aldrich) was used according to the manufacturer Several mycoparasites have been identified as bio- instructions to extract total RNA. The quality of RNA was control agents against plant phytoparasitic fungi. checked on 1% agarose and proceeded for cDNA Trichoderma harzianum is a soil borne and non- synthesis using RevertAid™ H Minus First strand cDNA pathogenic fungus that has been characterized for its synthesis kit (Thermos Scientific). The chitinase gene mycoparasitic activities [5, 6]. Mycoparasitism is the sequence submitted in NCBI GenBank (accession # defense mechanism present in fungi, in which one ARJ31758.1) was used to design forward 5′- fungus kill the other fungus [7]. During this complex CGCCTCGACGCCAGCTTTCTG-3′ and reverse 5′- defense mechanism, a variety of inhibitory proteins are CGCTGTCGCCAAGTGTCCAGTTA-3′ primer for secreted including ribosome-inactivating proteins (RIP), amplification. PCR reaction was carried out in thermal antimicrobial peptide and pathogenesis related (PR) cycler (ABI; 9700 system). The cycling profile consisted proteins like chitinases and gluconates which have of amplification reaction with following parameters: 5 min strong activity against fungal pathogens. The initial denaturation at 95ºC followed by 35 cycles with mycoparasitism potential of T. harzianum has been denaturation at 95ºC for 30 sec, annealing at 61ºC for 30 extensively investigated as bio-fungicide to control sec and extension at 72ºC for 45 sec followed by final Fusarium graminearum [8], Alternaria alternate, extension at 72ºC for 7 min. The amplicon of the resulting Fusarium oxysporum [9], Rhizoctonia solani [10] and the PCR products was detected on 1% agarose and cloned root-knot nematode Meloidogyne javanica [11]. Several into T-A end of pCR2.1 vector followed by transformation investigations have focused on the usage and in E. coli top10 cells. Positive clones were confirmed by manipulation of chitinase genes to enhance a plant’s restriction digestion and sequencing. ability to resist fungal pathogens. As an extracellular enzyme, chitinase hydrolyzes the chitin present in the Bioinformatic Analysis of Trichoderma harzianum cell wall of the phyto-pathogenic fungi. Recently, genes chitinase which encode chitinase are being cloned from various The sequence was compared with known sequences in microorganisms including Bacillus NCBI database using the NCBI BLAST server circulans [12], Streptomyces thermophiles [13], (http://www.ncbi.nlm.gov/BLAST). For the prediction of and Serratia marcescens [14] for heterologous TM helix, The Predict protein function was used expression in E. coli. Heterologous expression of several (https://ppopen.informatik.tu-muenchen.de/) and different proteins in Pichia pastoris (methylotrophic structural function was predicted by CFSSP Server – yeast) has been reported as it has certain advantages Chou & Fasman Secondary Structure Prediction Server over E. coli. It is cost effective, gave higher expression (http://www.biogem.org/tool/chou-fasman). The levels and have ability to perform many post-translational molecular weights and theoretical pIs were predicted modifications specific to eukaryotes including using the Compute pI/Mw tool glycosylation, proteolytic processing, disulfide bond (https://web.expasy.org/compute_pi/). DeepLoc-1.0 formation and minimal protein misfolding [15, 16]. (http://www.cbs.dtu.dk/services/DeepLoc/) was Agricultural crops suffer significant yield losses of combined to predict the subcellular localization of the about 26-30% from fungal pathogens and constitute proteins. Possible protein-polynucleotide binding sites serious threat to global food security. The incorporation was predicted from https://open.predictprotein.org/. for of inhibitory protein genes like chitinases in economically the analysis of hydrophobicity or hydrophilicity of protein, important crop plant will reduce the growth of pathogen, Protscale (http://web.expasy.org/protscale/) was used hence increasing tolerance against fungal pathogens. In and to predict the presence and location of signal this study, we cloned T. harzianum cDNA chitinase, peptide cleavage sites. SignalP 4.1 als Advancements in Life Sciences | www.als-journal.com | May 2020 | Volume 7 | Issue 3 123 You’re reading Molecular cloning and expression of recombinant Trichoderma harzianum chitinase in Pichia pastoris (http://www.cbs.dtu.dk/services/SignalP/) was continuous shaking at 250 rpm. Further, grown culture employed. Domain was identified with Interpro was induced with 0.5% methanol (v/v) and samples were (https://www.ebi.ac.uk/interpro/) and scanprosite taken at 24 hr, 48 hr, 72 hr and 96 hr. Desalting https://prosite.expasy.org/scanprosite) was used to chromatography (Amersham pharmacia) was performed identified specific active site. The sequence were align with concentrated supernatant, equilibrated with 20mM by muscle algorithm and was used to construct Tris–HCl buffer (pH 8.0). Purification of recombinant phylogenetic trees using MEGA-X. chitinase protein fractions was done by anion-exchange chromatography Source 30Q (Amersham pharmacia). Construction of recombinant pPICZ-chiF construct The purified protein was dialyzed against 20mM sodium and Transformation of Pichia pastoris GS115