02422nas a2200253   4500008004100000022001400041245009200055210006900147260000800216300001100224490000800235520158900243653002201832653002301854653002301877653002001900653003701920100001801957700002001975700002601995700001702021700001802038856011202056       2020                        eng d  a1369-703X00aUnderstanding unfolding and refolding of the antibody fragment (Fab). I. In-vitro study0 aUnderstanding unfolding and refolding of the antibody fragment F  cDEC  a1077640 v1643 a<p>In-vitro protein refolding is a major rate-limiting step in the large scale production of antibody fragments expressed using a microbial source like E. coli. This investigation is focused on understanding the in-vitro unfolding and refolding of the multi-domain protein involving inter-domain disulfide linkage, like antibody fragment (Fab). Solubilization behavior of the inclusion bodies and unfolding events of Fab fragment (Biosimilar rHu Ranibizumab) were studied using nano-differential scanning fluorimetry (nano-DSF). Fab unfolding behavior was studied by fitting experimental data with the two-state and three-state thermodynamic model. Based on the Fab unfolding understanding, a two-stage design of experiment (DoE) strategy was used for the optimization of the in-vitro refolding condition of a Fab fragment. Refolding yield of 56.03 +/- 1.15 % was achieved using the optimized oxidative refolding conditions maintained by appropriate dilution factor and redox reagent ratio. Refolding kinetics of the rHu Ranibizumab was analyzed using a three-parameter kinetic model showing rate constant k(1) :7.05e(-6) l/mg.min, k(2) :0.57 l/mg.min, and k(3) :310.19 l/mg.min. Based on observed refolding kinetics, it was concluded that the Fab refolding follows a three-state mechanism with the refolding intermediate/(s) formation from light and heavy chain of the Fab fragment as an overall rate-limiting step. The method described here is a useful tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.</p>
10aAntibody fragment10aIn-vitro refolding10aRefolding kinetics10arHu Ranibizumab10aTwo-state and Three-state models1 aGani, Kayanat1 aBhambure, Rahul1 aDeulgaonkar, Prashant1 aMehta, Deepa1 aKamble, Manoj  uhttp://library.ncl.res.in/content/understanding-unfolding-and-refolding-antibody-fragment-fab-i-vitro-study02418nas a2200253   4500008004100000022001400041245015400055210006900209260000800278300001100286490000800297520148800305653002501793653002901818653003501847653003101882653002501913100001701938700002001955700002401975700001801999700002002017856012702037       2021                        eng d  a1369-703X00aCloning and expression of antibody fragment (Fab) I: effect of expression construct and induction strategies on light and heavy chain gene expression0 aCloning and expression of antibody fragment Fab I effect of expr  cDEC  a1081890 v1763 aDual promoter expression constructs offer time and cost-effective alternatives to produce multi-domain proteins like antibody fragments. This investigation is focused on understanding the effect of expression construct (dual promoter vs. co-transformation strategy), codon optimization, and induction strategies on yield and expression stoichiometry of LC and HC genes of antibody fragment at shake-flask and bioreactor scale. rHu biosimilar Ranibizumab was selected as a model protein for the study. Expression stoichiometry of HC and LC gene at mRNA level was studied using RTqPCR, whereas protein expression level was studied quantitatively using RP-HPLC and SDS-PAGE analysis. In the case of dual promoter expression construct, it was observed that LC gene cloned in the MCS1 of the duet vectors has > 2-fold expression than the HC gene, cloned in the MCS2. Transcript abundance profile of the HC and LC genes determined at different time intervals post-induction shows a difference in the gene expression at the transcriptional level. Comparative analysis of dual promoter and co-transformation strategy shows better stoichiometry in co-transformation (1:1.3), whereas higher protein yield in a dual expression system (>2.4 fold). The use of lactose and galactose as inducers show higher Fab yield of 2.30 +/- 0.03 g/L and 2.81 +/- 0.06 g/L with expression stoichiometry of 1:1.9 and 1:2 (HC: LC) respectively than IPTG-based induction with a protein yield of 1.40 +/- 0.02 g/L.10aDual promoter vector10aExpression stoichiometry10aHigh cell density fermentation10arHu biosimilar Ranibizumab10aTranscript abundance1 aMehta, Deepa1 aChirmade, Tejas1 aTungekar, Aatir, A.1 aGani, Kayanat1 aBhambure, Rahul  uhttp://library.ncl.res.in/content/cloning-and-expression-antibody-fragment-fab-i-effect-expression-construct-and-induction02177nas a2200217   4500008004100000245011100041210006900152260000800221300001200229490000800241520143400249100001801683700001501701700002001716700001801736700002101754700001701775700002001792700002001812856012701832       2022                        eng d00aDimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-190 aDimerization of SARSCoV2 nucleocapsid protein affects sensitivit  cMAR  a428-4370 v2003 a<p>Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.</p>
1 aKhan, Wajihul1 aKhan, Nida1 aMishra, Avinash1 aGupta, Surbhi1 aBansode, Vikrant1 aMehta, Deepa1 aBhambure, Rahul1 aRathore, Anurag  uhttp://library.ncl.res.in/content/dimerization-sars-cov-2-nucleocapsid-protein-affects-sensitivity-elisa-based-diagnostics