Talaromyces leycettanus JCM12802 (Japan Collection of Microorganisms RIKEN BioResource Center, Tsukuba, Japan) was cultured in medium containing lignocellulose as the sole carbon source at 45 °C for 3 days [32 (link)]. Escherichia coli strain Trans I-T1 and the pEASY-T3 vector (TransGen, Beijing, China) were employed for DNA manipulation. P. pastoris GS115 and the pPIC9 vector (Invitrogen, Carlsbad, CA, USA) were used for heterologous gene expression.
Ppic9 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PPIC9 vector is a plasmid DNA vector designed for cloning and expressing recombinant proteins in various cell lines. The vector contains the necessary genetic elements, such as a promoter, multiple cloning sites, and selection markers, to facilitate the insertion and expression of target genes. The core function of the PPIC9 vector is to provide a standardized platform for the production of recombinant proteins in a laboratory setting, without making any claims about its intended use or applications.
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Lab products found in correlation
Peasy t3 vector, by Transgene (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
Qiaprep, by Qiagen (1 mentions)
Aluminum hydroxide alum, by Merck Group (1 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Phenyl sepharose 6ff column, by GE Healthcare (1 mentions)
Escherichia coli bl21 de3, by Transgene (1 mentions)
Pet22b, by Thermo Fisher Scientific (1 mentions)
Restriction endonuclease, by Takara Bio (1 mentions)
Beechwood xylan, by Merck Group (1 mentions)
Total rna isolation system kit, by Promega (1 mentions)
Dna purification kit, by Takara Bio (1 mentions)
La taq dna polymerase, by Takara Bio (1 mentions)
T4 dna ligase, by Promega (1 mentions)
8 protocols using ppic9 vector
1
Lignocellulose Degradation by Talaromyces
2
Recombinant Production of ChtI Enzyme Mutants
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The gene fragment encoding OfChtI-CAD (residues 19–407) was amplified from the full-length cDNA of OfChtI (GenBank ID DQ294305) with the primers 5′-TGAAGCTTACGTAGAATTC GCGGAGTCGGACAGCAGAGCG-3′ (forward) and 5′-CCGCCCTAGGGAATTC TTAATGATGATGATGATGATGAGAACGCGGTGGTGGAACAG-3′ (reverse). EcoRI restriction sites (bold) and a C-terminal 6×His affinity tag were introduced. The resulting PCR fragment was ligated into the pPIC9 vector (Invitrogen, Carlsbad, Califonia, USA) with an in-fusion HD cloning kit (Clontech, Palo Alto, California, USA).
The OfChtI-CAD E148Q, E148A, F159A, F194A, W241A, Y290A, F194A/W241A, F159A/Y290A and F159A/F194A/W241A/Y290A mutants were produced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California, USA), according to the manufacturer’s instructions. The mutated genes were sequenced to confirm that the desired mutations had been inserted.
The OfChtI-CAD E148Q, E148A, F159A, F194A, W241A, Y290A, F194A/W241A, F159A/Y290A and F159A/F194A/W241A/Y290A mutants were produced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California, USA), according to the manufacturer’s instructions. The mutated genes were sequenced to confirm that the desired mutations had been inserted.
3
Constructing pPic9abaecin Plasmid
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The insert was removed from vector pUC57 using the NotI and EcoRI enzymes. The vector pPic9 was also digested with the same enzymes, quantified and dephosphorylated with SAP enzyme. It was used 16.7 ng of the insert in the ligation reaction with 50 ng of pPIC9 vector and 1U of the DNA ligase I (Invitrogen) at 14 °C overnight. The product of this reaction was named pPic9abacin, confirmed by electrophorese at 0.8% of agarose. The Escherichia coli Top10 bacteria were transformed with the pPic9abaecin by electroporation in 0.2 cm cuvette with lysogeny broth (LB), following the parameters: 2.5 kV, 200 Ω and 25 µF, electroporated in Bio-Rad Gene Pulser (Bio-Rad). These clones were cultivated in LB medium containing 50 µg/mL of Ampicillin. The plasmid extraction was performed using QIAprep® (Qiagen®), following the kit protocol.
4
Influenza Virus Propagation and Inactivation
5
Expression and Purification of M2e5x Protein
6
Recombinant X24 Protein Expression in Pichia
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The X24 gene from P. cryptogea with α-secretion factor (Saccharomyces cerevisiae) was inserted into a pPIC9 vector (Life Technologies, USA). In addition, a glycine residue was added on the N-terminus of the recombinant protein in order to improve the processing ability of the KEX2 protease (α-secretion factor cleavage). A QuikChange kit (Stratagene, France) for direct mutagenesis was then used to construct the desired expression vectors by using oligonucleotides listed in Supplementary Table S1 . All vectors were validated by DNA sequencing. The constructed vectors were transformed into Pichia pastoris strain GS115. Screening for optimal protein production was performed and the most suitable strain was cultivated in a Biostat B-DCU bioreactor (Sartorius, Germany) using a previously described protocol (Wood and Komives, 1999 (link)). The expressed proteins were purified by ultrafiltration and Fast Protein Liquid Chromatography using a Source S15 column as described before (Pleskova et al., 2011 (link)). The molecular weights (MW) of the purified proteins were determined by MALDI-TOF spectroscopy.
7
Protein Expression in E. coli and P. pastoris
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Escherichia coli BL21 (DE3) (Transgen, China) was used as the host for prokaryotic protein expression. It was grown aerobically in Luria–Bertani broth with 100 µg of ampicillin/mL. The vector pET-22b(+) and shuttle vector pPIC9 (Invitrogen, China) were used for secretion of target proteins. All enzymes were purchased from Takara (Japan). Primers and genes were synthesised by Tsingke Biotech (China). P. pastoris GS115 (Invitrogen, Beijing, China) was used as the host for eukaryotic protein expression. The culture media were: minimal dextrose (MD), yeast extract peptone dextrose (YPD), buffered glycerol-complex (BMGY) and buffered methanol-complex (BMMY). All chemical reagents were of analytical pure grade.
8
Cloning and Expression of Xylanase in Pichia pastoris
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T. leycettanus JCM12802 was grown in potato dextrose Broth (PDB). Escherichia coli Trans1-T1 was used for gene cloning. Vector pPIC9 (Invitrogen, Carlsbad, CA) was used for gene expression by being transformed into Pichia pastoris GS115. Beechwood xylan was purchased from Sigma-Aldrich (St. Louis, MO). The DNA purification kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa (Otsu, Japan). The total RNA isolation system kit and T4 DNA ligase were purchased from Promega (Madison, WI). All chemicals were of analytical grade and commercially available.
+ Expand
T. leycettanus JCM12802 was grown in potato dextrose Broth (PDB). Escherichia coli Trans1-T1 was used for gene cloning. Vector pPIC9 (Invitrogen, Carlsbad, CA) was used for gene expression by being transformed into Pichia pastoris GS115. Beechwood xylan was purchased from Sigma-Aldrich (St. Louis, MO). The DNA purification kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa (Otsu, Japan). The total RNA isolation system kit and T4 DNA ligase were purchased from Promega (Madison, WI). All chemicals were of analytical grade and commercially available.
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