Kozlovskaia T M, Pushko P M, Stankevich E I, Dreĭmane A Ia, Sniker D Ia
Mol Biol (Mosk). 1988 May-Jun;22(3):731-40.
Expression of the coat protein gene of RNA bacteriophage fr in Escherichia coli cells leads to the formation of capsid-like structures of ca. 25 nm in diameter, which are immunologically indistinguishable from the native phage fr capsids. The modification strategy of the coat protein gene by gene engineering technique was developed in order to localize coat protein regions, which are exposed on the capsid surface and are capable to include foreign amino acid inserts without an appreciable effect on the capsid self-assembly. The oligonucleotide linkers, coding short amino acid sequences and bearing also convenient restriction sites, were synthesized and inserted into different regions of the coat protein gene. The mutant proteins, containing insertions of 2-12 amino acids in potentially exposed regions, were obtained. It was shown that N- and C-terminal insertions, as well as the insertion into codon 51 in the RNA-binding region, do not prevent the self-assembly. The regions (codons 96 and 112) were also revealed, insertions in them decreased drastically the protein yield as a consequence of a block in the self-assembly.
RNA噬菌体fr的外壳蛋白基因在大肠杆菌细胞中的表达导致形成直径约25nm的衣壳样结构，这些结构在免疫学上与天然噬菌体fr衣壳无法区分。为了定位衣壳蛋白暴露在衣壳表面且能够包含外源氨基酸插入片段而对衣壳自组装没有明显影响的区域，开发了通过基因工程技术对衣壳蛋白基因进行修饰的策略。合成了编码短氨基酸序列并带有方便的限制酶切位点的寡核苷酸接头，并将其插入衣壳蛋白基因的不同区域。获得了在潜在暴露区域含有2至12个氨基酸插入的突变蛋白。结果表明，N端和C端插入以及在RNA结合区域的密码子51处的插入并不妨碍自组装。还发现了一些区域（密码子96和112），在这些区域中的插入由于自组装受阻而大幅降低了蛋白质产量。
