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MALS驗證的高生物活性重組三聚體蛋白

背景
具有正確折疊和構象的重組蛋白才能有效保證抗體藥物研發的前期免疫、抗體篩選和鑒定、候選抗體藥物體外功能驗證、抗體藥物質量控制以及臨床樣本分析等過程的順利進行。蛋白質只有在正確的聚體結構下才能有效激活下游通路,發揮其生理功能。比如,在調控免疫應答過程中發揮重要的作用的腫瘤壞死因子(TNF)超家族,該系列蛋白在天然狀態下為三聚體結構,只有當三聚體形式TNF-α與TNFR1的胞外區結合時才能引起TNFR1的三聚化,進而有效的激發下游通路引起細胞凋亡。蛋白質的聚集狀態是決定其功能和活性的重要性質之一,我們不僅要在重組蛋白設計、表達和生產過程中嚴謹以待,還需要建立準確可靠的分子量測定分析方法來表征蛋白的聚集狀態。

圖1.SEC-MALS檢測原理圖
a.光散射示意圖 b.分析物分子量計算公式
常見的蛋白分子量測定技術有相對定量法(SEC-HPLC,高效分子篩液相色譜)和絕對定量法(SEC-MALS,多角度激光散射),兩種技術都可以測定溶液中蛋白的分子量,但由于原理上的不同,導致檢測結果準確度存在顯著差異(表1)。SEC-HPLC需要借助標準蛋白進行分子量內插測定,實際應用中常常受到多種因素的干擾,無法準確得到蛋白質真實分子量和聚集狀態。MALS技術是基于蛋白的分子量與光散射的強度直接相關來測定蛋白絕對分子量的技術,無需使用標準蛋白,不依賴于洗脫體積。通過SEC分離后的各組分進入多角光散射檢測器,激光照射到分析物時會發生光的散射,MALS通過多個角度同時測定散射光的強度(圖1.a),公式b1可以看出散射光強度正比于摩爾質量、濃度、折光指數增量的平方,且通過用公式b2可以直接計算出分析物的絕對分子量。
為滿足市場需求,ACROBiosystems已經開發了21種由HEK293表達的經過MALS驗證的TNF超家族分子蛋白,ACRO提供的TNF系列蛋白適用于大分子抗體藥物的免疫、SPR、細胞活性檢測等實驗,更有AvitagTM生物素標記的蛋白可以助力于您的藥物開發。
表1. SEC-HPLC和SEC-MALS兩種檢測方法的比較
檢測方法 原理和特點 結果
SEC-HPLC 柱校正法
  • 假設待測蛋白與標準蛋白的構象,形狀相似
  • 假設與柱填料無任何非特異的相互作用
  • 假設每次洗脫時間可重現無漂移
通過比較待測蛋白和標準蛋白的洗脫保留時間內插計算相對分子量
SEC-MALS 光散射法
  • 散射光強度正比于摩爾質量、濃度、折光指數增量的平方
  • 散射光角度依賴性僅與分子尺寸大小相關
通過激光散射強度直接測定絕對分子量,分子量測定不依賴于洗脫體積或保留時間
產品特點
SEC-MALS驗證蛋白的真實分子量和聚體狀態
OXL-H52Q8
Human OX40 Ligand /TNFSF4 Protein, His Tag, active trimer

Fig.1 The purity of Human OX40 Ligand, His Tag, active trimer (Cat. No. OXL-H52Q8) was more than 95% and around 60-80 kDa verified by SEC-MALS.

Protocol

CDL-H5266
Human CD27 Ligand / CD70 Protein, Fc Tag, active trimer

Fig.2 The purity of Human CD27 Ligand, Fc Tag, active trimer (MALS verified, Cat. No. CDL-H5266) was more than 85% and around 160-190 kDa verified by SEC-MALS.

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蛋白的生物活性由細胞實驗、SPR、ELISA多種技術的驗證
CDL-H5269
Human CD40 Ligand / TNFSF5 Protein, Fc Tag, active trimer

Fig.3 FACS analysis shows that Human CD40 Ligand, Fc Tag, active trimer (MALS verified) (Cat. No. CDL-H5269) can bind to 293T-CD40 cells surface CD40. The concentration of Human CD40 Ligand is 0.3 μg/mL.

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TNA-H4211
ActiveMax? Human TNF-alpha Protein, Tag Free, active trimer

Fig.4 ActiveMax? Human TNF-alpha, Tag Free, low endotoxin (active trimer) (MALS verified) (Cat. No. TNA-H4211) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.007-0.014 ng/mL.

Protocol

41L-H52D4
Human 4-1BB Ligand / TNFSF9 Protein, His,Flag Tag, active trimer

Fig.5 Human 4-1BB Ligand (71-254), His,Flag Tag, active trimer (MALS verified) (Cat. No. 41L-H52D4) induce IL-8 secretion in HT1080 human CD137 cell line. The EC50 for this effect is 0.20-0.44 μg/mL.

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LIT-H5242
Human LIGHT /TNFSF14 Protein, His Tag, active trimer

Fig.6 Human LIGHT, His Tag, active trimer (MALS verified) (Cat. No. LIT-H5242) induced cytotoxicity in HT-29 cells. The ED50 for this effect is 1.11-3.87 ng/mL.国产视频app

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RAL-H5240
Human TNFSF11 /RANKL /CD254 Protein, His Tag, active trimer

Fig.7 Captured Human RANK, Mouse IgG2a Fc Tag, low endotoxin (Cat. No. RAK-H5251) on CM5 chip via Anti-Mouse antibodies surface can bind Human TNFSF11, His Tag, active trimer (Cat. No. RAL-H5240) with an affinity constant of 0.309 nM as determined in a SPR assay (Biacore T200).

Protocol

CDL-H5266
Human CD27 Ligand / CD70 Protein, Fc Tag, active trimer

Fig.8 Immobilized Human CD27, His Tag (Cat. No. CD7-H522b) at 2 μg/mL (100 μL/well) can bind Human CD27 Ligand, Fc Tag, active trimer (MALS verified) (Cat. No. CDL-H5266) with a linear range of 0.2-8 ng/mL.

Protocol

產品列表
MALS驗證的TNF超家族產品列表
Molecule Cat. No. Species Product Description
4-1BB Ligand 41L-H5265 Human Human 4-1BB Ligand / TNFSF9 (71-254) Protein, Fc Tag (active trimer) (MALS verified)
41L-H52D4 Human Human 4-1BB Ligand / TNFSF9 (71-254) Protein, His,Flag Tag (active trimer) (MALS verified)
BAFF BAF-H82F3 Human Biotinylated Human BAFF / TNFSF13B / CD257 Protein, Avitag?,Fc Tag (active trimer) (MALS verified)
BAF-H82Q2 Human Biotinylated Human BAFF / TNFSF13B / CD257 Protein, His,Avitag? (active trimer) (MALS verified)
CD27 Ligand CDL-H5266 Human Human CD27 Ligand / CD70 Protein, Fc Tag (active trimer) (MALS verified)
CDL-H52Da Human Human CD27 Ligand / CD70 Protein, His,Flag Tag (active trimer) (MALS verified)
CDL-H82D7 Human Biotinylated Human CD27 Ligand / CD70 Protein, His,Avitag?,Flag Tag (active trimer) (MALS verified)
CDL-M5245 Mouse Mouse CD27 Ligand / CD70 Protein, His Tag (active trimer) (MALS verified)
CD40 Ligand CDL-H5269 Human Human CD40 Ligand / TNFSF5 Protein, Fc Tag (active trimer) (MALS verified)
CDL-H52Db Human Human CD40 Ligand / TNFSF5 Protein, His,Flag Tag (active trimer) (MALS verified)
CDL-M5248 Mouse Mouse CD40 Ligand / TNFSF5 Protein, His Tag (active trimer) (MALS verified)
Fas Ligand FAL-H5241 Human Human Fas Ligand / TNFSF6 Protein, His Tag (active trimer) (MALS verified)
LIGHT LIT-H5242 Human Human LIGHT / TNFSF14 Protein, His Tag (active trimer) (MALS verified)
LIT-H5269 Human Human LIGHT / TNFSF14 Protein, Fc Tag (active trimer) (MALS verified)
OX40 Ligand OXL-H52Q8 Human Human OX40 Ligand / TNFSF4 Protein, His Tag (active trimer) (MALS verified)
TNF-alpha TNA-H4211 Human ActiveMax® Human TNF-alpha Protein, Tag Free, low endotoxin (active trimer) (MALS verified)
TNA-H5228 Human Human TNF-alpha Protein, His Tag (active trimer) (MALS verified)
TNA-H8211 Human Biotinylated Human TNF-alpha Protein, epitope tag free, ultra sensitivity (primary amine labeling) (active trimer) (MALS verified)
TNA-H82E3 Human Biotinylated Human TNF-alpha Protein, His,Avitag? (active trimer) (MALS verified)
TNA-M82E9 Mouse Biotinylated Mouse TNF-alpha Protein, His,Avitag? (active trimer) (MALS verified)
TNFSF11 RAL-H5240 Human Human TNFSF11 / RANKL / CD254 Protein, His Tag (active trimer) (MALS verified)
MALS驗證的其他天然三聚體蛋白
Molecule Cat. No. Species Product Description
S protein SPN-C52H8 2019-nCoV 2019-nCoV (COVID-19) Full Length S protein (R683A, R685A) , His Tag, active trimer (MALS verified)
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