Cloning and expression of two genes coding endo-β-1,4-glucanases from Trichoderma asperellum PQ34 in Pichia pastoris

2016 ◽  
Vol 70 (3) ◽  
Author(s):  
Nguyen Hoang Loc ◽  
Le My Tieu Ngoc ◽  
Hoang Tan Quang ◽  
Nguyen Duc Huy ◽  
Nguyen Ngoc Luong
Keyword(s):  
Pichia Pastoris ◽  
Signal Peptide ◽  
Induction Time ◽  
Culture Medium ◽  
Cloning And Expression ◽  
Trichoderma Asperellum ◽  
Glucanase Activity ◽  
Inducer Concentration ◽  
Molecular Masses

AbstractTwo genes coding endo-β-1,4-glucanases were cloned from Trichoderma asperellum PQ34 which was isolated from Thua Thien Hue province, Vietnam. The expression of these genes in Pichia pastoris produced two enzymes with molecular masses of approximately 46 kDa (about 42 kDa of enzymes and 4 kDa of signal peptide). The effects of induction time and temperature, inducer concentration, and culture medium on the endo-β-1,4-glucanase activity were investigated. The results showed that the highest total activities of two endo-β-1,4-glucanases were approximately 4.7 × 10

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

Cloning, Expression, and Characterization of Mouse Tissue Factor Pathway Inhibitor (TFPI)

1998 ◽  
Vol 79 (02) ◽  
pp. 306-309 ◽  
Author(s):  
Dougald Monroe ◽  
Julie Oliver ◽  
Darla Liles ◽  
Harold Roberts ◽  
Jen-Yea Chang
Keyword(s):  
Amino Acid ◽  
Tissue Factor ◽  
Signal Peptide ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Mouse Tissue ◽  
Amino Acid Residues ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

Cloning and Expression of a Novel Xylanase Gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris

2012 ◽  
Vol 167 (8) ◽  
pp. 2198-2211 ◽  
Author(s):  
Huimin Zhang ◽  
Minchen Wu ◽  
Jianfang Li ◽  
Shujuan Gao ◽  
Yanjun Yang
Keyword(s):  
Pichia Pastoris ◽  
Cloning And Expression ◽  
Xylanase Gene ◽  
Aspergillus Usamii

scholarly journals Characterization of the ToxB Gene from Pyrenophora tritici-repentis

2001 ◽  
Vol 14 (5) ◽  
pp. 675-677 ◽  
Author(s):  
J. Patrick Martinez ◽  
Sean A. Ottum ◽  
Shaukat Ali ◽  
Leonard J. Francl ◽  
Lynda M. Ciuffetti
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Heterologous Expression ◽  
North Dakota ◽  
Signal Peptide ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Pyrenophora Tritici Repentis ◽  
Putative Signal Peptide

The ToxB gene was cloned and characterized from a race 5 isolate of Pyrenophora tritici-repentis from North Dakota. ToxB contains a 261-bp open reading frame that encodes a 23 amino acid putative signal peptide and a 64 amino acid host-selective toxin, Ptr ToxB. Analysis of Ptr ToxB from heterologous expression in Pichia pastoris confirms that ToxB encodes a host-selective toxin.

scholarly journals Compatibility of Trichoderma isolates with pesticides used in lettuce crop

2018 ◽  
Vol 44 (2) ◽  
pp. 137-142 ◽  
Author(s):  
Micaele Aparecida Franco da Silva ◽  
Karina Elaine de Moura ◽  
Kamila Ellen de Moura ◽  
Denise Salomão ◽  
Flávia Rodrigues Alves Patricio
Keyword(s):  
Biological Control ◽  
Mycelial Growth ◽  
Culture Medium ◽  
Conidial Germination ◽  
The Other ◽  
Trichoderma Asperellum ◽  
Sclerotinia Minor ◽  
Other Hand ◽  
Lettuce Drop ◽  
Trichoderma Isolates

ABSTRACT Lettuce drop, caused by Sclerotinia minor and S. sclerotiorum, is one of the most important diseases that affect lettuce crop in Brazil. In previous studies, isolates of Trichoderma asperellum (IBLF 897, IBLF 904 and IBLF 914) and T. asperelloides (IBLF 908) were selected for the biocontrol of this disease. In this subsequent study, the compatibility of these isolates with pesticides used in lettuce crop in Brazil was evaluated. Initially, the mycelial growth of isolates was evaluated in culture medium plus pesticides. Then, the effect of pesticides on the parasitism of T. asperelloides isolate IBLF 914 in baits and sclerotia of S. minor and S. sclerotiorum, as well as on the survival of lettuce seedlings, was evaluated in gerboxes after application on baits and sclerotia of the antagonist with pesticides at their respective commercial doses. The fungicides pencycuron and mandipropamid and the insecticide imidacloprid did not affect the mycelial growth of Trichoderma isolates. The fungicide iprodione did not affect the mycelial growth of T. asperellum isolates. but the isolate of T. asperelloides was sensitive from the concentration of 10 µg.L-1 fungicide. Procymidone reduced the mycelial growth of Trichoderma isolates from the concentration of 10 µg.L-1 fungicide, and azoxystrobin reduced the conidial germination of the isolates of the antagonist, showing LD50 between 0.36 and 0.42 µg.L-1 fungicide. On the other hand, in the experiment carried out in “gerboxes”, none of the pesticides reduced the parasitism of baits and sclerotia or reduced the control of S. minor and S. sclerotiorum in lettuce seedlings. Results indicate that the biological control of lettuce drop with T. asperelum isolate IBLF 914 can be compatible with the remaining phytosanitary treatments used in lettuce crop.

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