Specific binding of immunoglobulin G to protein A–mesoporous silica composites for affinity column chromatography

2013 ◽  
Vol 1 (45) ◽  
pp. 6321 ◽  
Author(s):  
Kazuma Nakanishi ◽  
Masahiro Tomita ◽  
Hitomi Nakamura ◽  
Katsuya Kato
Keyword(s):  
Mesoporous Silica ◽  
Column Chromatography ◽  
Immunoglobulin G ◽  
Specific Binding ◽  
Protein A ◽  
Affinity Column ◽  
Affinity Column Chromatography

scholarly journals Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Balamurugan Shanmugaraj ◽  
Kaewta Rattanapisit ◽  
Suwimon Manopwisedjaroen ◽  
Arunee Thitithanyanont ◽  
Waranyoo Phoolcharoen
Keyword(s):  
Monoclonal Antibodies ◽  
Nicotiana Benthamiana ◽  
Specific Binding ◽  
Expression System ◽  
Protein A ◽  
Single Step ◽  
Affinity Column ◽  
Plant Expression ◽  
Plant Expression System

The ongoing coronavirus disease 2019 (COVID-19) outbreak caused by novel zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially reported in Wuhan city, Hubei Province of China, in late December 2019. The rapid global spread of the virus calls for the urgent development of vaccines or therapeutics for human applications to combat the coronavirus infection. Monoclonal antibodies (mAbs) have been utilized as effective therapeutics for treating various infectious diseases. In the present study, we evaluated the feasibility of plant expression system for the rapid production of recently identified therapeutically suitable human anti-SARS-CoV-2 mAbs B38 and H4. Transient co-expression of heavy-chain and light-chain sequences of both the antibodies by using plant expression geminiviral vector resulted in rapid accumulation of assembled mAbs in Nicotiana benthamiana leaves within 4 days post-infiltration. Furthermore, both the mAbs were purified from the plant crude extracts with single-step protein A affinity column chromatography. The expression level of mAb B38 and H4 was estimated to be 4 and 35 μg/g leaf fresh weight, respectively. Both plant-produced mAbs demonstrated specific binding to receptor binding domain (RBD) of SARS-CoV-2 and exhibited efficient virus neutralization activity in vitro. To the best of our knowledge, this is the first report of functional anti-SARS-CoV-2 mAbs produced in plants, which demonstrates the ability of using a plant expression system as a suitable platform for the production of effective, safe, and affordable SARS-CoV-2 mAbs to fight against the spread of this highly infectious pathogen.

scholarly journals Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

2006 ◽  
Vol 399 (2) ◽  
pp. 325-333 ◽  
Author(s):  
In-Ra Seo ◽  
Sang Hyun Moh ◽  
Eun Hui Lee ◽  
Gerhard Meissner ◽  
Do Han Kim
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ryanodine Receptor ◽  
Column Chromatography ◽  
Anion Channel ◽  
Affinity Column ◽  
Open Probability ◽  
Release Channel ◽  
Affinity Column Chromatography ◽  
Lifetime Analysis ◽  
Associated Proteins

DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean Po (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 μM DIDS induced reversible long-lived open events (Po=0.451±0.038) in the presence of 10 μM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 μM DIDS became considerably less potent (Po=0.206±0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.

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