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An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution.

Tabuchi I, Soramoto S, Suzuki M, Nishigaki K, Nemoto N, Husimi Y - Biol Proced Online (2002)

Bottom Line: This reaction gave a yield of about 95%.We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity.An efficient amplification of the in vitro virus with this "viral genome" was demonstrated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Functional Materials Science, Saitama University. 255 Shimo-okubo, Saitama 338-857. Japan.; GenCom Co. 11 Minami-Oya, Machida 194-8511. Japan.Present address: Tokyo Evolution Research Center. 1-1-45-504, Okubo, Shinjuku-ku, Tokyo, 169-0072. Japan. husimi@fms.saitama-u.ac.jp

ABSTRACT
The "in vitro virus" is a molecular construct to perform evolutionary protein engineering. The "virion (=viral particle)" (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this "viral genome" was demonstrated.

No MeSH data available.


Related in: MedlinePlus

The dependence of the Y-ligation efficiency on the molar ratio, [Linker-Y]/ [mRNA]. Concentration of mRNA was [mRNA] = 0.5 µM. The ligation efficiency is defined as the final fraction (at incubation time 3,600s) of the ligation product.
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Figure 4: The dependence of the Y-ligation efficiency on the molar ratio, [Linker-Y]/ [mRNA]. Concentration of mRNA was [mRNA] = 0.5 µM. The ligation efficiency is defined as the final fraction (at incubation time 3,600s) of the ligation product.

Mentions: The dependence of the ligation efficiency on the molar ratio [mRNA]:[Linker-Y] is shown in Fig. 4. The Y-ligation reaction proceeded with a high efficiency, even in a 1:1 ratio. Moreover, the removal of excess linkers was not necessary.


An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution.

Tabuchi I, Soramoto S, Suzuki M, Nishigaki K, Nemoto N, Husimi Y - Biol Proced Online (2002)

The dependence of the Y-ligation efficiency on the molar ratio, [Linker-Y]/ [mRNA]. Concentration of mRNA was [mRNA] = 0.5 µM. The ligation efficiency is defined as the final fraction (at incubation time 3,600s) of the ligation product.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC145556&req=5

Figure 4: The dependence of the Y-ligation efficiency on the molar ratio, [Linker-Y]/ [mRNA]. Concentration of mRNA was [mRNA] = 0.5 µM. The ligation efficiency is defined as the final fraction (at incubation time 3,600s) of the ligation product.
Mentions: The dependence of the ligation efficiency on the molar ratio [mRNA]:[Linker-Y] is shown in Fig. 4. The Y-ligation reaction proceeded with a high efficiency, even in a 1:1 ratio. Moreover, the removal of excess linkers was not necessary.

Bottom Line: This reaction gave a yield of about 95%.We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity.An efficient amplification of the in vitro virus with this "viral genome" was demonstrated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Functional Materials Science, Saitama University. 255 Shimo-okubo, Saitama 338-857. Japan.; GenCom Co. 11 Minami-Oya, Machida 194-8511. Japan.Present address: Tokyo Evolution Research Center. 1-1-45-504, Okubo, Shinjuku-ku, Tokyo, 169-0072. Japan. husimi@fms.saitama-u.ac.jp

ABSTRACT
The "in vitro virus" is a molecular construct to perform evolutionary protein engineering. The "virion (=viral particle)" (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this "viral genome" was demonstrated.

No MeSH data available.


Related in: MedlinePlus