Cloning and expression of antibody fragment (Fab) I: Effect of expression construct and induction strategies on light and heavy chain gene expression was written by Mehta, Deepa;Chirmade, Tejas;Tungekar, Aatir A.;Gani, Kayanat;Bhambure, Rahul. And the article was included in Biochemical Engineering Journal in 2021.Safety of (2R,3R,4S,5R,6S)-2-(Hydroxymethyl)-6-(isopropylthio)tetrahydro-2H-pyran-3,4,5-triol The following contents are mentioned in the article:
Dual 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 quant. using RP-HPLC and SDS-PAGE anal. 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 anal. 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) resp. than IPTG-based induction with a protein yield of 1.40 ± 0.02 g/L. This study involved multiple reactions and reactants, such as (2R,3R,4S,5R,6S)-2-(Hydroxymethyl)-6-(isopropylthio)tetrahydro-2H-pyran-3,4,5-triol (cas: 367-93-1Safety of (2R,3R,4S,5R,6S)-2-(Hydroxymethyl)-6-(isopropylthio)tetrahydro-2H-pyran-3,4,5-triol).
(2R,3R,4S,5R,6S)-2-(Hydroxymethyl)-6-(isopropylthio)tetrahydro-2H-pyran-3,4,5-triol (cas: 367-93-1) belongs to alcohols. Alcohols are weak acids. The most acidic simple alcohols (methanol and ethanol) are about as acidic as water, and most other alcohols are somewhat less acidic. Alcohols may be oxidized to give ketones, aldehydes, and carboxylic acids. These functional groups are useful for further reactions. Oxidation of organic compounds generally increases the number of bonds from carbon to oxygen (or another electronegative element, such as a halogen), and it may decrease the number of bonds to hydrogen.Safety of (2R,3R,4S,5R,6S)-2-(Hydroxymethyl)-6-(isopropylthio)tetrahydro-2H-pyran-3,4,5-triol
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