P. pastoris strain GS115 and expression vector pPIC9 were from Invitrogen (Grand Island, NY). S. aureus strains SA113 and S. aureus subsp. aureus (ATCC 25923) were obtained from the American Type Culture Collection (Manassas, VA). Other clinical isolates of S. aureus (methicillin sensitive strains 6445 and 3425-1, and MRSA strain 3425-3) were the kind gift of Dr Ambrose Cheung (Dartmouth, Hanover, NH).
P pastoris strain gs115
Manufactured by Thermo Fisher Scientific
Sourced in United States
P. pastoris strain GS115 is a yeast strain commonly used in the laboratory for the expression of recombinant proteins. It is a methylotrophic yeast, capable of utilizing methanol as a sole carbon source. The GS115 strain is a histidine auxotroph, requiring the addition of histidine for growth.
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Lab products found in correlation
Ppic9, by Thermo Fisher Scientific (2 mentions)
Ppic9k, by Thermo Fisher Scientific (2 mentions)
Ppicza, by Thermo Fisher Scientific (1 mentions)
E coli dh5α, by Takara Bio (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Sac 1, by Thermo Fisher Scientific (1 mentions)
Ecori, by Thermo Fisher Scientific (1 mentions)
Yeast extract peptone dextrose medium, by Thermo Fisher Scientific (1 mentions)
Escherichia coli strain top10, by Promega (1 mentions)
Pgem t, by Promega (1 mentions)
Escherichia coli strain dh5α competent cells, by Takara Bio (1 mentions)
Ppic9k vector, by Thermo Fisher Scientific (1 mentions)
E coli top10, by Thermo Fisher Scientific (1 mentions)
Herdchek csfv ab elisa kit, by IDEXX (1 mentions)
7 protocols using p pastoris strain gs115
1
Expression of Staphylococcus Strains
2
Heterologous Protein Expression in P. pastoris
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The strains, plasmids, and primers used in this study are listed in Table 1 . E. coli DH5α, purchased from Takara (Dalian, China), was used for plasmid proliferation. P. pastoris strain GS115, two yeast expression vectors, pPIC9K and pPICZA, as well as all restriction enzymes (EcoRI, NotI, XhoI, SalI, SacI, and PmeI) were purchased from Invitrogen (Carlsbad, CA, USA).
Luria-Bertani (LB) medium contained 10 g/L sodium chloride, 10 g/L tryptone, and 5 g/L yeast extract. The yeast extract peptone dextrose (YPD) medium contained 20 g/L glucose, 20 g/L tryptone, and 10 g/L yeast extract. The minimal dextrose (MD) medium contained 13.4 g/L yeast nitrogen base (YNB), 4 × 10−4 g/L biotin, and 20 g/L glucose. The buffered glycerol-complex (BMGY) medium contained 20 g/L tryptone, 10 g/L yeast extract, 13.4 g/L yeast nitrogen base (YNB), 4 × 10−4 g/L biotin, 100 mmol/L potassium phosphate buffer (pH 6.0), and 10 g/L glycerol. Buffered methanol-complex (BMMY) medium contained 20 g/L tryptone, 10 g/L yeast extract, 13.4 g/L yeast nitrogen base (YNB), 4 × 10−4 g/L biotin, 100 mmol/L potassium phosphate buffer (pH 6.0), and 5 g/L methanol.
Luria-Bertani (LB) medium contained 10 g/L sodium chloride, 10 g/L tryptone, and 5 g/L yeast extract. The yeast extract peptone dextrose (YPD) medium contained 20 g/L glucose, 20 g/L tryptone, and 10 g/L yeast extract. The minimal dextrose (MD) medium contained 13.4 g/L yeast nitrogen base (YNB), 4 × 10−4 g/L biotin, and 20 g/L glucose. The buffered glycerol-complex (BMGY) medium contained 20 g/L tryptone, 10 g/L yeast extract, 13.4 g/L yeast nitrogen base (YNB), 4 × 10−4 g/L biotin, 100 mmol/L potassium phosphate buffer (pH 6.0), and 10 g/L glycerol. Buffered methanol-complex (BMMY) medium contained 20 g/L tryptone, 10 g/L yeast extract, 13.4 g/L yeast nitrogen base (YNB), 4 × 10−4 g/L biotin, 100 mmol/L potassium phosphate buffer (pH 6.0), and 5 g/L methanol.
3
Recombinant Protein Expression Protocols
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The GenBank accession numbers of Ochrobactrum sp. M231 MPH and OPHC2 are ACC63894 and CAE53631, respectively. Escherichia coli strain Top10 and the plasmid pGEM-T were purchased from Promega Corp. (Madison, WI, USA). E. coli strain BL21(DE3) and the expression plasmid pET-30a(+) were purchased from Novagen (Darmstadt, Germany). P. pastoris strain GS115 and the vector pPIC9 were purchased from Invitrogen (Carlsbad, CA).
E. coli cells were cultured aerobically at 37°C in Luria-Bertani medium. Minimal dextrose (MD) medium, buffered complex glycerol medium (BMGY), yeast extract peptone dextrose medium (YPD), and buffered complex menthol medium (BMMY) were prepared according to the manufacturer’s instructions (Invitrogen).
E. coli cells were cultured aerobically at 37°C in Luria-Bertani medium. Minimal dextrose (MD) medium, buffered complex glycerol medium (BMGY), yeast extract peptone dextrose medium (YPD), and buffered complex menthol medium (BMMY) were prepared according to the manufacturer’s instructions (Invitrogen).
4
Isolation and Expression of T. marneffei from AIDS Patient
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T. marneffei strain GD-0079 was isolated from the bone marrow of an AIDS patient with the complication of penicilliosis. The strain was confirmed via bone marrow culture at Guangzhou Eighth People's Hospital and maintained at the Research Center for Pathogenic Fungi within the hospital. The GD-0079 isolate was inoculated on Sabouraud dextrose agar (Difco, BD, Baltimore, MD, USA) at 25 °C as mycelia and converted to yeast at 37 °C.
The P. pastoris expression kit containing pPIC9K vectors and P. pastoris strain GS115 were purchased from Invitrogen Corp. (Carlsbad, CA, USA). The Escherichia coli strain DH5α-competent cells were purchased from Takara (Dalian, China). E. coli cells with plasmids were cultured at 37 °C in Luria–Bertani medium (5 g/L yeast extract, 10 g/L tryptone,10 g/L NaCl and 15 g/L agar) containing 100 μg/mL ampicillin to maintain the plasmids. Minimal dextrose (MD) medium, yeast extract-peptone-dextrose (YPD) medium, buffered complex glycerol (BMGY) medium and buffered complex methanol (BMMY) medium were prepared according to the instructions of Invitrogen for P. pastoris fermentation.
The P. pastoris expression kit containing pPIC9K vectors and P. pastoris strain GS115 were purchased from Invitrogen Corp. (Carlsbad, CA, USA). The Escherichia coli strain DH5α-competent cells were purchased from Takara (Dalian, China). E. coli cells with plasmids were cultured at 37 °C in Luria–Bertani medium (5 g/L yeast extract, 10 g/L tryptone,10 g/L NaCl and 15 g/L agar) containing 100 μg/mL ampicillin to maintain the plasmids. Minimal dextrose (MD) medium, yeast extract-peptone-dextrose (YPD) medium, buffered complex glycerol (BMGY) medium and buffered complex methanol (BMMY) medium were prepared according to the instructions of Invitrogen for P. pastoris fermentation.
5
Recombinant FhCystatin Protein Production
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The FhCystatin gene was subcloned into the pPIC9K vector (Invitrogen, San Diego, California, USA) to generate the pPIC9K-FhCystatin recombinant plasmid, and positive clones were screened using PCR and restriction digestion. Then, pPIC9K-FhCystatin was linearized with Sal I and electrotransformed into P. pastoris GS115 strain (Invitrogen) to screen the recombinant yeast strain. The recombinant yeast was cultured in BMGY medium and continuously induced with methanol (1%) for 5 day. Subsequently, the cells were precipitated by centrifugation and the culture supernatant was collected for SDS-PAGE analysis. Homogeneity of rFhCystatin was examined by 15% reducing SDS-PAGE. Western blot analysis was performed using Fh-infected sheep serum as primary antibody (dilution ratio 1:1,000) and horseradish peroxidase-conjugated rabbit anti-sheep IgG (Abcam, Cambridge, UK) as secondary antibody (dilution ratio 1:3,000). rFhCystatin was purified using Ni-NTA chromatography (Merck, Darmstadt, Germany) according to the manufacturer’s instructions.
6
Bacterial and Yeast Cultivation Protocols
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The S. aureus strain (ATCC6538) was cultivated in LB medium. The E. coli Top10 (Invitrogen, Carlsbad, CA, USA) was used for vector manipulation and propagation. The E. coli DE3(BL21)/pTRX-srtA [27 ], was constructed in our laboratory and cultivated in LB medium, which was added ampicillin at 100 μg/mL as a marker. The P. pastoris GS115 strain (Invitrogen, Carlsbad, CA, USA) and the yeast-displayed vector pKFS [28 (link)] were used to display the LPETG-EGFPs. P. pastoris GS115 was cultivated in MD (1.34% (w/v) yeast nitrogen base, 6 × 10−5% biotin, 1% (w/v) dextrose), YPD (1% yeast extract, 2% peptone, 2% dextrose), BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% Biotin, 1% glycerol) and BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% Biotin, 1% methanol) medium.
7
Evaluating CSFV Antibody Detection Assays
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The yeast expression vector pPIC9K and the yeast P. pastoris GS115 strain were purchased from Invitrogen (Carlsbad, USA).
The following serum samples were tested in this study: (1) 254 swine serum samples confirmed to be free of CSFV antibodies using the virus neutralization test (VNT) and the HerdChek CSFV Ab ELISA kit (IDEXX, USA) for cutoff value determination; (2) sequential serum samples from 9 pigs upon vaccination with CSFV C-strain or marker vaccine rAdV-SFV-E2, and subsequent challenge with CSFV virulent Shimen strain; (3) a panel of swine sera for comparison of yE rns -iELISA with VNT, including 63 sera from pigs experimentally infected with the CSFV Shimen strain, 18 C-strain-vaccinated swine sera, 34 rAdV-SFV-E2-immunized swine sera and 101 clinical swine serum samples.
Other serum samples included 3 sera from pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV), 3 sera from pigs experimentally infected with pseudorabies virus (PRV), 3 sera from pigs infected with porcine circovirus type 2 (PCV2), 3 sera from pigs experimentally infected with porcine epidemic diarrhea virus (PEDV), 19 sera from pigs experimentally infected with BVDV (4 sera from the EU Reference Laboratory for CSF, Hannover, Germany) and 4 sera from pigs experimentally infected with BDV (from the EU Reference Laboratory for CSF).
The following serum samples were tested in this study: (1) 254 swine serum samples confirmed to be free of CSFV antibodies using the virus neutralization test (VNT) and the HerdChek CSFV Ab ELISA kit (IDEXX, USA) for cutoff value determination; (2) sequential serum samples from 9 pigs upon vaccination with CSFV C-strain or marker vaccine rAdV-SFV-E2, and subsequent challenge with CSFV virulent Shimen strain; (3) a panel of swine sera for comparison of yE rns -iELISA with VNT, including 63 sera from pigs experimentally infected with the CSFV Shimen strain, 18 C-strain-vaccinated swine sera, 34 rAdV-SFV-E2-immunized swine sera and 101 clinical swine serum samples.
Other serum samples included 3 sera from pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV), 3 sera from pigs experimentally infected with pseudorabies virus (PRV), 3 sera from pigs infected with porcine circovirus type 2 (PCV2), 3 sera from pigs experimentally infected with porcine epidemic diarrhea virus (PEDV), 19 sera from pigs experimentally infected with BVDV (4 sera from the EU Reference Laboratory for CSF, Hannover, Germany) and 4 sera from pigs experimentally infected with BDV (from the EU Reference Laboratory for CSF).
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