02419nas a2200253   4500008004100000022001400041245015400055210006900209260000800278300001100286490000800297520148800305653002501793653002901818653003501847653003101882653002501913100001701938700002001955700002401975700001801999700002002017856012802037       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  uhttps://library.ncl.res.in/content/cloning-and-expression-antibody-fragment-fab-i-effect-expression-construct-and-induction03440nas a2200313   4500008004100000245016600041210006900207260000800276300000800284490000700292520240600299653000902705653001602714653002502730653002402755653001302779653001002792653000802802653002402810100002002834700002002854700001902874700002102893700001902914700002202933700002002955700002402975856012702999       2022                        eng d00aComparative analysis and structural modeling of elaeis oleifera FAD2, a fatty acid desaturase involved in unsaturated fatty acid composition of american oil palm0 aComparative analysis and structural modeling of elaeis oleifera   cAPR  a5290 v113 a<p>
	Simple Summary Palm oil has become the world's most important vegetable oil in terms of production quantity, and its overall demand is exponentially growing with the global population. The fatty acid composition and particularly the oleic/linoleic acid ratio are major factors influencing palm oil quality. In this study, we focused on FAD2, a fatty acid desaturase enzyme involved in the desaturation and conversion of oleic acid to linoleic acid in Elaeis oleifera, identified through in silico annotation analysis. Our phylogenetic and comparative studies revealed two SNP markers, SNP278 and SNP851, significantly correlated with the oleic/linoleic acid contents. Our study provides fundamental insights into the mechanism of fatty acids synthesis in oil palm and could support the application of molecular biology techniques to enhance the enzymatic activity and substrate affinity of EoFAD2. American oil palm (Elaeis oleifera) is an important source of dietary oil that could fulfill the increasing worldwide demand for cooking oil. Therefore, improving its production is crucial and could be realized through breeding and genetic engineering approaches aiming to obtain high-yielding varieties with improved oil content and quality. The fatty acid composition and particularly the oleic/linoleic acid ratio are major factors influencing oil quality. Our work focused on a fatty acid desaturase (FAD) enzyme involved in the desaturation and conversion of oleic acid to linoleic acid. Following the in silico identification and annotation of Elaeis oleifera FAD2, its molecular and structural features characterization was performed to better understand the mechanistic bases of its enzymatic activity. EoFAD2 is 1173 nucleotides long and encodes a protein of 390 amino acids that shares similarities with other FADs. Interestingly, the phylogenetic study showed three distinguished groups where EoFAD2 clustered among monocotyledonous taxa. EoFAD2 is a membrane-bound protein with five transmembrane domains presumably located in the endoplasmic reticulum. The homodimer organization model of EoFAD2 enzyme and substrates and respective substrate-binding residues were predicted and described. Moreover, the comparison between 24 FAD2 sequences from different species generated two interesting single-nucleotide polymorphisms (SNPs) associated with the oleic/linoleic acid contents.</p>
10aFAD210aFatty acids10ain silico annotation10alinoleic acid ratio10aoil palm10aoleic10aSNP10astructural modeling1 aBen Ayed, Rayda1 aChirmade, Tejas1 aHanana, Mohsen1 aKhamassi, Khalil1 aErcisli, Sezai1 aChoudhary, Ravish1 aKadoo, Narendra1 aKarunakaran, Rohini  uhttps://library.ncl.res.in/content/comparative-analysis-and-structural-modeling-elaeis-oleifera-fad2-fatty-acid-desaturase02165nas a2200229   4500008004100000022001400041245012600055210006900181260000800250490000800258520133400266653002401600653002901624653002201653653003101675653002501706100001701731700002001748700002001768700002001788856012701808       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  uhttps://library.ncl.res.in/content/cloning-and-expression-antibody-fragment-fab-ii-effect-expression-hosts-light-and-heavy