Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-1β synthesis in Escherichia coli. / Mashko, Sergey V.; Mochulsky, Andrey V.; Kotenko, Sergey V.; Lebedeva, Marina I.; Lapidus, Alla L.; Mochulskaya, Natalya A.; Izotova, Lara S.; Veiko, Vladimir P.; Vinetsky, Yury P.; Ketlinsky, Sergey A.; Debabov, Vladimir G.
In: Gene, Vol. 97, No. 2, 15.01.1991, p. 259-266.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-1β synthesis in Escherichia coli
AU - Mashko, Sergey V.
AU - Mochulsky, Andrey V.
AU - Kotenko, Sergey V.
AU - Lebedeva, Marina I.
AU - Lapidus, Alla L.
AU - Mochulskaya, Natalya A.
AU - Izotova, Lara S.
AU - Veiko, Vladimir P.
AU - Vinetsky, Yury P.
AU - Ketlinsky, Sergey A.
AU - Debabov, Vladimir G.
PY - 1991/1/15
Y1 - 1991/1/15
N2 - A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121-126]. In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and alow Cop at28°C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42°C. Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-lβ (hIL-1β) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1β(re-hIL-1β) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the watersoluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1β. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3-5 mg ofre-hIL-lβ/g of wet cells. The re-hIL-1β specific activity was about 2 × 108 units/mg, coinciding with that of the authentic hIL-1β.
AB - A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121-126]. In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and alow Cop at28°C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42°C. Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-lβ (hIL-1β) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1β(re-hIL-1β) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the watersoluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1β. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3-5 mg ofre-hIL-lβ/g of wet cells. The re-hIL-1β specific activity was about 2 × 108 units/mg, coinciding with that of the authentic hIL-1β.
KW - overlappon
KW - p and p promoters of λ phage
KW - plasmid copy number
KW - protein purification
KW - Recombinant DNA
KW - TGATG vector
UR - http://www.scopus.com/inward/record.url?scp=0026013407&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(91)90060-O
DO - 10.1016/0378-1119(91)90060-O
M3 - Article
C2 - 1999290
AN - SCOPUS:0026013407
VL - 97
SP - 259
EP - 266
JO - Gene
JF - Gene
SN - 0378-1119
IS - 2
ER -
ID: 90132810