Abstract
We describe an improved method for the production of recombinant Qβ replicase heterotetramer. The successful expression of the soluble Qβ RNA polymerase complex depends on the EF-Ts and EF-Tu subunits being co-expressed prior to β-subunit expression. Efficient co-expression requires two different inducible operons to co-ordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex is achieved by production of the recombinant S1-subunit of Qβ replicase in a separate host. This approach represents a facile way for producing and purifying large amounts of soluble and active recombinant Qβ replicase tetramer without the necessity of a His-tag for purification.
| Language | English |
|---|---|
| Pages | 651-659 |
| Number of pages | 9 |
| Journal | Applied Biochemistry and Biotechnology |
| Volume | 169 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jan 2013 |
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Facile production and rapid purification of functional recombinant Qβ replicase heterotetramer complex. / Gunasekaran, Karthikeyan; Bergquist, Peter L.; Sunna, Anwar.
In: Applied Biochemistry and Biotechnology, Vol. 169, No. 2, 01.2013, p. 651-659.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Facile production and rapid purification of functional recombinant Qβ replicase heterotetramer complex
AU - Gunasekaran, Karthikeyan
AU - Bergquist, Peter L.
AU - Sunna, Anwar
PY - 2013/1
Y1 - 2013/1
N2 - We describe an improved method for the production of recombinant Qβ replicase heterotetramer. The successful expression of the soluble Qβ RNA polymerase complex depends on the EF-Ts and EF-Tu subunits being co-expressed prior to β-subunit expression. Efficient co-expression requires two different inducible operons to co-ordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex is achieved by production of the recombinant S1-subunit of Qβ replicase in a separate host. This approach represents a facile way for producing and purifying large amounts of soluble and active recombinant Qβ replicase tetramer without the necessity of a His-tag for purification.
AB - We describe an improved method for the production of recombinant Qβ replicase heterotetramer. The successful expression of the soluble Qβ RNA polymerase complex depends on the EF-Ts and EF-Tu subunits being co-expressed prior to β-subunit expression. Efficient co-expression requires two different inducible operons to co-ordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex is achieved by production of the recombinant S1-subunit of Qβ replicase in a separate host. This approach represents a facile way for producing and purifying large amounts of soluble and active recombinant Qβ replicase tetramer without the necessity of a His-tag for purification.
UR - http://www.scopus.com/inward/record.url?scp=84873094273&partnerID=8YFLogxK
U2 - 10.1007/s12010-012-0018-9
DO - 10.1007/s12010-012-0018-9
M3 - Article
VL - 169
SP - 651
EP - 659
JO - Applied Biochemistry and Biotechnology
T2 - Applied Biochemistry and Biotechnology
JF - Applied Biochemistry and Biotechnology
SN - 0273-2289
IS - 2
ER -