Soluble GP proteins were expressed using the ExpiCHO-S expression system (ThermoFisher Scientific) and in ExpiCHO-S Expression Medium (GibcoTM). This expression system comprises mammalian Chinese Hamster Ovary (11 (link)) cells, the most commonly used expression system for the production of biotherapeutics (12 (link)). We hypothesize that CHO-based expression of recombinant fusion proteins will result in a glycosylation profile reflecting that of the native viral protein following infection. Transfection was conducted following the manufacturer’s protocol for transient expression (ThermoFisher Scientific) using plasmid DNA at a ratio of 1 µg of DNA per 1 ml of culture volume when the culture cell density was 6 × 106 cells/ml. Five to 7 days after transfection, the cells from each culture flask was pelleted following centrifugation (3,500 g at 4°C for 10 min) and the culture supernatant (SN) was sterile-filtered (0.22 µm) to perform protein purifications.
Expicho s expression system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ExpiCHO-S expression system is a cell line and media system designed for the high-yield production of recombinant proteins in Chinese Hamster Ovary (CHO) cells. It is a complete solution for the transient expression of proteins in CHO suspension cultures.
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Lab products found in correlation
Pcag neo, by Fujifilm (1 mentions)
Pcag ble, by Fujifilm (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Normal human igg, by Merck Group (1 mentions)
Pcag ble vector, by Fujifilm (1 mentions)
Pcag neo vector, by Fujifilm (1 mentions)
Hb a0, by Merck Group (1 mentions)
Ni nta superflow column, by Qiagen (1 mentions)
4 protocols using expicho s expression system
1
Recombinant Viral Glycoprotein Expression
2
Generation of cancer-specific anti-PDPN antibody
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PMab-38, a mouse anti-dPDPN mAb (ImmunoglobulinG1 (IgG1), kappa), was developed as previously described [15 (link)]. To generate P38B, a mouse-canine (subclass B) chimeric antibody, the appropriate VH and VL cDNAs of mouse PMab-38 and the CH and CL of canine IgG subclass B were subcloned into pCAG-Ble and pCAG-Neo vectors (FUJIFILM Wako Pure Chemical Corporation) [36 (link)]. To generate a cancer-specific mouse–dog chimeric anti-PDPN antibody (P38Bf), antibody expression vectors were transfected into BINDS-09 (FUT8-knocked out ExpiCHO-S cells; http://www.med-tohoku-antibody.com/topics/001_paper_cell.htm ) using the ExpiCHO-S Expression System (Thermo Fisher Scientific, Waltham, MA, USA). P38Bf was purified using Protein G-Sepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
3
Humanization of Anti-PDPN Monoclonal Antibody
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Normal human IgG was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The complementarity determining region (CDR) of LpMab-23 VH, frame sequences of VH in human Ig, and CH of human IgG1 were cloned into the pCAG-Neo vector [FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan] to generate a humanized anti-human PDPN mAb (humLpMab-23). The CDR of LpMab-23 VL, frame sequences of VL in human Ig, and CL of human kappa chain were cloned into the pCAG-Ble vector (Wako). We transfected the antibody expression vectors of humLpMab-23 into ExpiCHO-S or BINDS-09 cells using the ExpiCHO-S Expression System (Thermo Fisher Scientific, Inc.). We named these mAbs humLpMab-23 and humLpMab-23-f, respectively. We purified humLpMab-23 and humLpMab-23-f using Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan).
4
Haptoglobin Phenotyping and Purification
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In total, 112 anonymized plasma samples were collected from surplus material from patient samples at the Department of Clinical Biochemistry and Pharmacology, Odense University Hospital in the period between September 2018 and November 2018. The study was conducted as a quality study with anonymous blood samples, which according to the legislation under the Danish Health Authorities does not require approval from ethical committee. The study was approved by the Region of Southern Denmark (reference number 18/51099). Samples were stored at -80 o C until further analysis. Hp phenotype was determined by western blotting. The samples showed the following distribution Hp1-1 (n=17, 15.2%), Hp2-1 (n=53, 47.3%) and Hp2-2 (n=42, 37.5%).
Native purified Hp1-1 and Hp2-2 from human serum were purchased (#H0138; #H9762; Sigma-Aldrich). Recombinant Hp1-1 (rHp1-1, NP_001119574.1) and recombinant Hpr (rHpr, AAA88081.1) were expressed using the ExpiCHO-S expression system (#A29133; ThermoFisher Scientific) according to manufacturer's instructions. rHp1-1 was expressed with a C-terminal His-Tag and was purified using a Ni-NTA Superflow column (#30430, Qiagen). On the other hand, rHpr was purified using Hb A0 (#H0267; Sigma-Aldrich) affinity chromatography.
Native purified Hp1-1 and Hp2-2 from human serum were purchased (#H0138; #H9762; Sigma-Aldrich). Recombinant Hp1-1 (rHp1-1, NP_001119574.1) and recombinant Hpr (rHpr, AAA88081.1) were expressed using the ExpiCHO-S expression system (#A29133; ThermoFisher Scientific) according to manufacturer's instructions. rHp1-1 was expressed with a C-terminal His-Tag and was purified using a Ni-NTA Superflow column (#30430, Qiagen). On the other hand, rHpr was purified using Hb A0 (#H0267; Sigma-Aldrich) affinity chromatography.
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