Goriunova L E, Sakharova N K, Barbakar' N I, Grechko V V
Mol Biol (Mosk). 1980 Sep-Oct;14(5):1173-86.
A wheat germ cell-free system was used for translation of kappa-chain IgG1 messenger RNA isolated from mouse plasmacytoma MOPC 21 cells polysomes. The system was optimised for a number of ingredients of the incubation mixture. Incorporation of labelled amino acids in polypeptides was shown as a function of K+, Mg2+, exogenous mRNA concentration and time, mRNA was purificated by two successive oligo (dT)-cellulose chromatographies and two successive sucrose gradient centrifugations or polyacrylamide gel electrophoresis. Different fractions of poly(A)-RNA stimulated protein synthesis 20--30-fold in a wheat germ cell-free system. Analysis of the translation product of 14 and 16S mRNA by SDS-polyacrylamide gel electrophoresis revealed polypeptide comigrating with the authentic L-chain IgG1 and specifically precipitating by an antiserum to mouse IgG and L-chain IgGl (MOPC 21). The 18 and 28S mRNA fractions directed the synthesis of polypeptides with molecular weight up to 50 000 dalton. Immunospecific translation product of the 18 and 28S mRNA contain both L- and H-chains.
利用小麦胚芽无细胞体系对从小鼠浆细胞瘤MOPC 21细胞多核糖体中分离得到的κ链IgG1信使核糖核酸（mRNA）进行翻译。该体系针对孵育混合物的多种成分进行了优化。以标记氨基酸掺入多肽的情况作为K⁺、Mg²⁺、外源mRNA浓度和时间的函数进行展示，mRNA通过两次连续的寡聚（dT）-纤维素柱层析以及两次连续的蔗糖梯度离心或聚丙烯酰胺凝胶电泳进行纯化。在小麦胚芽无细胞体系中，不同部分的多聚（A）-RNA可刺激蛋白质合成20至30倍。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对14S和16S mRNA的翻译产物进行分析，结果显示有一种多肽与天然IgG1轻链（L链）迁移率相同，且能被抗小鼠IgG和L链IgG1（MOPC 21）抗血清特异性沉淀。18S和28S mRNA部分指导合成分子量高达50000道尔顿的多肽。18S和28S mRNA的免疫特异性翻译产物包含轻链和重链。
