02418nas 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-induction02164nas a2200229   4500008004100000022001400041245012600055210006900181260000800250490000800258520133400266653002401600653002901624653002201653653003101675653002501706100001701731700002001748700002001768700002001788856012601808       2023                        eng d  a1369-703X00aCloning and expression of antibody fragment (Fab) II: Effect of expression hosts on light and heavy chain gene expression0 aCloning and expression of antibody fragment Fab II Effect of exp  cNOV0 v2003 a<p>Protein expression stoichiometry of individual subunits plays a critical role in the formation of multi-domain functional therapeutic proteins like antibody fragments. In this study, we have evaluated light to heavy chain ratio (LC: HC) of rHu Ranibizumab expressed using duet expression vector in six E. coli strains. The LC: HC ratio at the protein level was determined using RP-HPLC whereas transcript abundance of LC and HC at the mRNA level was determined using RTqPCR. Protein level study as well as the transcript abundance profiles of the LC and HC genes were observed to be differentially expressed across the E. coli strains. BL21 STAR (DE3) and Origami 2 strains resulted in a nearly equal expression ratio of LC and HC genes. Untargeted shotgun proteomics analysis was performed to identify the differentially expressed proteins. Total 1083 proteins were identified in the spectral IDA library, while 244 and 149 proteins were differentially expressed in Origami 2 and BL21 STAR (DE3) respectively. These proteins are associated with translation and nucleotide biosynthesis pathways. Ob-tained results provide insights into the impact of different E.coli host strains on LC: HC ratio, the probable reasons behind the unbalanced gene ratio, and provide useful transcriptomic and proteomics analysis protocols.</p>
10aE.coli host strains10aExpression stoichiometry10aProteome analysis10arHu biosimilar Ranibizumab10aTranscript abundance1 aMehta, Deepa1 aChirmade, Tejas1 aNainwal, Neeraj1 aBhambure, Rahul  uhttp://library.ncl.res.in/content/cloning-and-expression-antibody-fragment-fab-ii-effect-expression-hosts-light-and-heavy