In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

Bacteriophage GA coat protein formed self-assembly competent virus-like particles (VLPs) have been expressed previously in bacterial and yeast cells. On the basis of our previous experiments on the yeast vector pESC-URA / S. cerevisiae system containing two oppositely oriented promoters, new constructions were created with point-mutations in coat protein to mimic phage MS2-like RNA binding characteristics. Simultaneously, the MS2 operator sequence was added to mRNA desired for packaging. After the introduction of single-point mutations (S87N, K55N, R43K) and double-point mutations (S87N + K55N and S87N + R43K), the coat protein’s ability to form VLPs was retained, but yield from cells was decreased. Exchange of the 87^th Ser to Asn in coat protein sequence in combination with bacteriophage MS2 translational operator provided specific packaging of the gene of interest (GFP). Although non-specific nucleic acid sequences were packaged, the remarkable specificity for packaging of the gene of interest can be achieved using the described approach.