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HEK293 表達的Nanodisc / DDM全長CD20蛋白

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背景

Lionel Rougé, et al. Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab. Science. 2020
2020年2月20日Genentech在Science上發表的一篇文章Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab,突破性地解析了全長CD20蛋白以及CD20蛋白與Rituximab Fabs復合物的三維結構。文章證實Rituximab對CD20的識別不止有一級表位ECL2,還識別其二級表位ECL1/ECL2。SPR驗證的Rituximab與全長CD20蛋白以及與ECL2多肽表位的親和力差異,表明二級表位ECL1/ECL2會影響Rituximab和CD20的親和力。另外文章發現二級表位ECL1/ECL2還會影響Rituximab的CDC效應,揭示了具有完整結構的全長CD20蛋白的重要意義。(頁面尾部可下載文獻全文)
在2019年召開的第61屆美國血液學會(ASH)報道了CD20-CD3 T細胞雙特異性抗體CD20-TCB(RG6026)同時靶向B細胞表面的CD20和T細胞表面的CD3,其治療R/R B-NHL的2項臨床研究初步結果顯示該雙特異性抗體具有可接受的安全性以及有前景的療效。
CD20是淋巴癌、白血病和某些自身免疫等疾病治療的熱門目標靶點,其在免疫治療領域的應用非常廣泛。但因為全長CD20蛋白的表達難度高、活性不穩定等因素,目前市面上很難找到合適的CD20蛋白用于活性分析。
為了解決這些問題,ACROBiosystems采用獨特的設計開發了全球獨有的由HEK293表達的多次跨膜全長CD20蛋白,包括未進行標記的和生物素標記的Nanodisc或DDM不同版本產品。全長CD20蛋白同時有small loop和big loop,具有完整的天然正確構象,可用于免疫/ ELISA/ SPR/ BLI/ FACS。我們驗證了Rituximab、Ofatumumab與全長CD20蛋白的結合活性,全長CD20蛋白展現出納摩爾級別的高親和力,可免費提供Protocol。
不同類型的全長CD20蛋白
DDM版本
DDM是一種非離子型去垢劑,能夠使脂膜解體釋放膜蛋白,并在溶液中為去膜狀態下的膜蛋白提供疏水環境,維持和保護膜蛋白的疏水跨膜結構。但DDM的存在會破壞細胞膜的結構,一般不用推薦于細胞實驗。
Nanodisc版本(不含去垢劑)
Nanodisc (納米磷脂盤) 是由膜支架蛋白(membrane scaffold proteins, MSPs)和磷脂分子構成的磷脂雙分子層類膜結構,膜蛋白可以整合到Nanodisc的特殊結構中,保持其生物學活性并有很好的水溶性,應用更廣泛。ACROBiosystems使用的Nanodisc技術已經獲得專利持有方的授權,生物藥研發企業在研發過程中可放心使用。

Ilia G. Denisov, et al., Nanodisc in membrane biochemistry and biophysics. Chem Rev. 2017(頁面尾部可下載文獻全文)

產品列表
ACRO開發的生物素標記的全長CD20蛋白(DDM和Nanodisc)經FACS驗證具有高生物活性,點擊下方可申請protocol。
Molecule Cat. No. Host Type Product Description
CD20/ MS4A1 CD0-H52H1 HEK293 Unconjugated Human CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293)
Request FACS Protocol
CD0-H52H3 HEK293 Unconjugated Human CD20 / MS4A1 Full Length Protein, His Tag (HEK293) (SPR verified)
Request FACS Protocol
CD0-H82E3 HEK293 Biotin-labeled Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag? (Nanodisc) (HEK293)
Request FACS Protocol
CD0-H82E5 HEK293 Biotin-labeled Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag? (HEK293)
Request FACS Protocol
產品特點
全長CD20蛋白由真核系統表達具有完整的天然構象(同時具有small loop和big loop)
全長CD20蛋白能提供更好的活性

A) HEK293 Cell (Full Length)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-3 ng/mL (in presence of DDM and CHS).

Protocol

B) E.coli (Extracellular)

国产视频app

Immobilized Rituximab at 5 μg/mL (100 μL/well) can bind Human CD20, TrxA Tag, His Tag (E.coli) with a linear range of 78-2500 ng/mL.

ACRO的全長CD20蛋白活性經FACS/ELISA/ SPR驗證,可免費提供相應的Protocol
FACS驗證
Cat. No. CD0-H82E3 (HEK293表達)
FACS Analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) and negative control antibody respectively, washed and then followed by PE-SA and analyzed with FACS.

Protocol

Cat. No. CD0-H52H1 (HEK293表達)
FACS Analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Human CD20 Full Length, His Tag (Cat. No. CD0-H52H1) and negative control antibody respectively, washed and then followed by PE anti-His antibody and analyzed with FACS.

国产视频app Protocol

ELISA驗證
Cat. No. CD0-H82E3 (HEK293表達)

Immobilized Rituximab at 5 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) with a linear range of 0.008-0.125 μg/mL国产视频app

国产视频app Protocol

Cat. No. CD0-H52H1 (HEK293表達)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag (Cat. No. CD0-H52H1) with a linear range of 1-63 ng/mL

Protocol

Cat. No. CD0-H82E5 (HEK293表達)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4-63 ng/mL (in presence of DDM and CHS).国产视频app

国产视频app Protocol

Cat. No. CD0-H82E5 (HEK293表達)

Immobilized Ofatumumab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4-63 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H52H3 (HEK293表達)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-3 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H52H3 (HEK293表達)

Immobilized Ofatumumab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-6 ng/mL (in presence of DDM and CHS).

国产视频app Protocol

SPR驗證
Cat. No. CD0-H82E5 (HEK293表達)

Biotinylated Human CD20, His,Avitag (HEK293) (Cat. No. CD0-H82E5) captured on Biotin CAP-Series S Sensor Chip can bind Rituximab with an affinity constant of 1.73 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore T200).

国产视频app Protocol

Cat. No. CD0-H52H3 (HEK293表達)

Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) captured on CM5 chip via Anti-human IgG Fc antibodies surface can bind MabThera? (Rituximab) with an affinity constant of 6.21 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore 8K).国产视频app

Protocol

產品評論
 
評論(3)
  1. 158XXXXXXX9
  2. 18人贊
  3. BLI實驗。相比之前在另一家公司買的CD20蛋白,這個蛋白結合活性更強,CD20這種特殊膜蛋白,能夠全長表達還具有生物活性,還是可以的。就是價格能稍微便宜點就更好了
  4. 2019-10-23
  1. 150XXXXXXX0
  2. 13人贊
  3. CD20因為是多次跨膜蛋白,找了很多家公司都表達不好。 抗體篩選和Elisa都定了很多次,實驗效果不錯。 除了有點小貴沒有別的缺點啦!
  4. 2019-10-11
  1. 177XXXXXXX3
  2. 14人贊
  3. 重組抗CD20單克隆抗體的靶點受體是跨膜蛋白,且體外表達的CD20受體很難維持其生物活性,我們常采用細胞法或流式方法進行活性評價,其涉及細胞制備,周期比較長,重復性不是很好。因此我們迫切需要建立SPR及ELISA分析方法對重組抗CD20單克隆抗體結合活性進行評價,而這個體外表達的CD20受體蛋白正好滿足我們需求,并且已對其實用性進行驗證。對加快我門產品研發進度,非常有幫助!
  4. 2019-10-11
 
相關參考文獻

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab

Nanodisc in membrane biochemistry and biophysics.

  • Authors:国产视频app Ilia G. Denisov, et al.

  • Journal:国产视频app Chem Rev. 2017

  • Download Full Article

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