Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

Abstract

A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)+RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)'RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.

A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) inChlorella sorokiniana

“…1A). Because Bascomb et al [5] showed earlier that a 2.2-2.3 kb cDNA could be synthesized from highly purified C. sorokiniana N A D P -G D H mRNA, isolated by a very specific polysome immunoselection procedure, full-length N A D P -G D H cDNA was anticipated to be at least 2.2 kb. As described in a later section of this paper, northern blotting analyses showed the N A D P -G D H mRNA to be 2.2 kb.…”

How far are the new wave of mRNA drugs from us? mRNA product current perspective and future development

“…However, its synthetic design needs to be optimized to address its toxicity and tendency to aggregate and leak. The purity of mRNA has a significant impact on efficacy and safety, and efficient purification methods need to be developed to improve mRNA purity and remove impurities such as double-stranded RNA, truncated mRNA, and DNA residues ( 68 ). mRNA particle size uniformity can improve the stability of the LNP delivery system ( 69 ).…”

Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana

“…IA, lane 4). In a recent study, Bascomb et al (3) showed that in vitro translation of NADP-GDH mRNA, purified by a specific polysome immunoprecipitation procedure, yielded a single NADP-GDH antigen also with a mol wt of 58,500.…”