The recombinant plasmids pPIC9-XYL10C and pPIC9-XylE, which harbor the genes xyl10C (FJ492963) from Bispora sp. MEY-1 [13 (link)] and xylE (FJ860894) from P. canescens [22 (link)], were used in this work. Vectors pEASY-T3 and Escherichia coli Trans I-T1 were chosen for gene amplification. Pichia pastoris GS115 (Invitrogen, Carlsbad, CA) and pPIC9 (TransGen, Beijing, China) were used for protein expression. The cultivation and enzyme induction were conducted on the basis of the Pichia Expression kit instructions. The substrate beechwood xylan and standards were purchased from Sigma-Aldrich (St. Louis, MO). Restriction endonucleases, DNA polymerase, DNA purification kit, T4 DNA ligase, and endo-β-N-acetylglucosaminidase H (Endo H) were purchased from GenStar (Beijing, China). All other chemicals were of analytical grade.
Pichia pastoris gs115
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Pichia pastoris GS115 is a methylotrophic yeast strain commonly used for recombinant protein expression. It is a genetically modified version of the Pichia pastoris host, designed to facilitate high-level expression of heterologous proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ppic9k, by Thermo Fisher Scientific (4 mentions)
Ppic9, by Thermo Fisher Scientific (4 mentions)
Ppiczαa, by Thermo Fisher Scientific (3 mentions)
Escherichia coli trans1 t1, by Transgene (3 mentions)
Pichia expression kit, by Thermo Fisher Scientific (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
T4 dna ligase, by Takara Bio (3 mentions)
Beechwood xylan, by Merck Group (2 mentions)
Avicel ph 101, by Merck Group (2 mentions)
Gluconic acid, by Merck Group (2 mentions)
Ascorbate, by Merck Group (2 mentions)
Restriction endonuclease, by Takara Bio (2 mentions)
T4 dna ligase, by Promega (2 mentions)
Taq dna polymerase, by Takara Bio (2 mentions)
Pgapza, by Thermo Fisher Scientific (1 mentions)
Escherichia coli strain top10, by Thermo Fisher Scientific (1 mentions)
Pichia expression manual, by Thermo Fisher Scientific (1 mentions)
Pichia pastoris, by Thermo Fisher Scientific (1 mentions)
Manno oligosaccharides, by Megazyme (1 mentions)
Escherichia coli bl21, by Thermo Fisher Scientific (1 mentions)
36 protocols using pichia pastoris gs115
1
Recombinant Expression of Xylan-Degrading Enzymes
2
Heterologous Protein Expression in Pichia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli strain TOP10 was used for recombinant DNA manipulation. Pichia pastoris GS115 (Invitrogen) expressing eGFP or Phy was used as the wild-type (wt) strain and all deletion strains used in this study were derived from it. The pPICZαA (Invitrogen) vector including AOX1 promoter was used to express the heterologous protein of eGFP and Phy from Citrobacter amalonaticus CGMCC 1696 [57 (link)]. The pGAPZA (Invitrogen) vector containing GAP promoter was used to construct the RP complementation strains.
3
Heterologous Expression of P. oxalicum GZ-2 Enzyme
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The P. oxalicum GZ-2 referred to in this study was isolated and identified as previously reported [13 (link)] and has been deposited at the China General Microbiological Culture Collection Center (CGMCC 7527). Escherichia coli strain TOP 10 (Invitrogen, San Diego, CA) grown in Luria Bertani (LB) medium at 37°C was used as the host cell for plasmid cloning and propagation of the recombinant expression pPICZαA vector. Pichia pastoris GS115 (Invitrogen, San Diego, CA) was used for heterologous protein expression. All media and protocols for Pichia pastoris are described in the Pichia expression manual (Invitrogen, San Diego, CA). Manno-oligosaccharides were purchased from Megazyme (Bray, Ireland). P. oxalicum GZ-2 was maintained and cultured according previously study [32 (link)]. Pichia pastoris GS115 was cultivated at 30°C in yeast extract peptone dextrose (YPD) medium (1% yeast extract, 2% tryptone, and 2% dextrose).
4
Cultivating Fungal and Yeast Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The C. cinerea ATCC 56838 strain from the American Type Culture Collection (Manassas, VA, USA) was cultured for growth of fruiting bodies according to Zhang et al. [37 (link)].
Pichia pastoris GS115 from Invitrogen (San Diego, CA, USA) was cultivated for genetic manipulation according to the user manual provided by Invitrogen.
Pichia pastoris GS115 from Invitrogen (San Diego, CA, USA) was cultivated for genetic manipulation according to the user manual provided by Invitrogen.
5
Cloning and Expression of α-Amylase from Cystobacter sp.
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cloning of the α-amylase gene was performed using Cystobacter sp. strain CF23 as the source. The host strains utilized for gene cloning and protein expression were Escherichia coli DH5, Escherichia coli BL21 and Pichia pastoris GS115 (Invitrogen Corporation, Shanghai, China). The vector pEFαA was used for protein expression [3 (link)]. The starches obtained for the study were procured from Changhong Potato Starch Co., Ltd. (located in Hebei, China) and Yuanye Biotechnology Co., Ltd. (based in Shanghai, China). The Malto-oligosaccharides used in this study were sourced from Aladdin Co., Ltd. (Shanghai, China). The chemicals and reagents employed in this work were sourced from Sinopharm Chemical Reagent Co., Ltd. (located in Shanghai, China) and were of analytical grade.
6
Isolation and characterization of thermophilic fungus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chaetomium thermophilum CGMCC3.17990 strain was previously isolated in China and deposited in the publicly accessible culture collection CGMCC (Beijing, China). We purchased the plasmid vector pPICZαA and Pichia pastoris GS115 strain from Invitrogen. For total RNA isolation, we grew C. thermophilum at 50 °C for 48 h in a medium containing 2% avicel, 0.4% yeast extract, 0.1% K2HPO4·3H2O, and 0.05% MgSO4·7H2O, dissolved in tap water. Avicel PH-101, glucose, gluconic acid, and ascorbate were from Sigma-Aldrich. Cellodextrin oligosaccharide mixture and cellopentaose were from Elicityl (Crolles, France). Other reagents were of analytic grade.
7
Heterologous Expression of FaeA in Pichia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Insertion of the MtFae1a gene in pPNic706, transformation into Pichia pastoris GS115 (Invitrogen) and screening of P. pastoris transformants were performed by ProteoNic BV. (Leiden, the Netherlands), as described for Aspergillus niger FaeA in (Gidijala et al. 2018 (link)). Bioreactor fermentation for the production of P-Fae was performed as described in (Gidijala et al. 2018 (link)), except that the enzyme was harvested as follows. The protein-containing culture supernatant was separated from yeast cells by continuous high-speed centrifugation, and subsequently concentrated by crossflow filtration (10 kilo Daltons (kDa) cut-off, 4 °C). The resulting protein concentrate was lyophilized, ground and stored at − 20 °C until used for further experiments. P. pastoris is also known as Komagataella phaffii (Kurtzman 2009 (link)).
8
Recombinant protein production in Pichia pastoris
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli T1 (TransGen Biotech, Beijing, China) was used for gene cloning. Pichia pastoris GS115 (Invitrogen, Carlsbad, CA, USA) was used for recombinant protein production as a heterologous expression host. The Pichia-secreted expression vector pPIC9K (Invitrogen, Carlsbad, CA, USA) was used for heterologous expression. The pPIC9K/CTendo45 plasmid containing the β-1,4-endoglucanase gene CTendo45 (GenBank accession number KC441877) and a C-terminal 6× histidine tag sequence was prepared as described previously23 (link). The Fast Mutagenesis System Kit was purchased from TransGen Biotech (Beijing, China). Primers were synthesized by Sangon Biotech (Shanghai, China) and are listed in Supplementary Table S1 . All chemicals were of reagent grade purity.
9
Acquisition and Acclimation of Mature and Juvenile Micropterus salmoides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Firstly, the mature M. salmoides for the experiment were acquired from a marketplace in Hangzhou. The mean weight was 500 ± 10 g, while the body length averaged 23 ± 2 cm. Secondly, the juvenile M. salmoides were procured from a pisciculture facility in Chongqing. The juvenile M. salmoides exhibited an average weight of 1 ± 0.2 g and a body length of 5 ± 1 cm. Subsequently, the procured juveniles and mature M. salmoides were harbored in an aquarium within a piscatory laboratory for a week, ensuring their sound state of well-being, prior to their utilization as experimental specimens.
Micrococcus luteus and Escherichia coli were purchased from the Microbial Culture Collection Center (Beijing, China). V. parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus were purchased from the Global Biological Resource Center (United States). Pichia pastoris GS115 was from Invitrogen. A. hydrophila and Edwardsiella tarda were preserved by our laboratory.
Micrococcus luteus and Escherichia coli were purchased from the Microbial Culture Collection Center (Beijing, China). V. parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus were purchased from the Global Biological Resource Center (United States). Pichia pastoris GS115 was from Invitrogen. A. hydrophila and Edwardsiella tarda were preserved by our laboratory.
10
Recombinant Protein Production in Pichia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmid pPICZαA and Pichia pastoris GS115 were purchased from Invitrogen. Ascorbate, avicel PH-101, gluconic acid, galactose, sorbitol, and glucose were purchased from Sigma-Aldrich. Other chemicals were of analytical reagent grade and were obtained from Shandong Keshang Biotechnology (China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!