TGATG vector : a new expression system for cloned foreign genes in Escherichia coli cells. / Mashko, Sergey V.; Veiko, Vladimir P.; Lapidus, Alla L.; Lebedeva, Marina I.; Mochulsky, Andrey V.; Shechter, Inna I.; Trukhan, Maxim E.; Ratmanova, Ksenya I.; Rebentish, Boris A.; Kaluzhsky, Vasilii E.; Debabov, Vladimir G.
In: Gene, Vol. 88, No. 1, 30.03.1990, p. 121-126.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - TGATG vector
T2 - a new expression system for cloned foreign genes in Escherichia coli cells
AU - Mashko, Sergey V.
AU - Veiko, Vladimir P.
AU - Lapidus, Alla L.
AU - Lebedeva, Marina I.
AU - Mochulsky, Andrey V.
AU - Shechter, Inna I.
AU - Trukhan, Maxim E.
AU - Ratmanova, Ksenya I.
AU - Rebentish, Boris A.
AU - Kaluzhsky, Vasilii E.
AU - Debabov, Vladimir G.
PY - 1990/3/30
Y1 - 1990/3/30
N2 - A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage γ pR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage λ Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT'C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment. This system was successfully tested in experiments on the optimization of expression of the genes encoding human or interferons, human interleukin-1β and E. coli β-galactosidase in E. coli cells.
AB - A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage γ pR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage λ Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT'C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment. This system was successfully tested in experiments on the optimization of expression of the genes encoding human or interferons, human interleukin-1β and E. coli β-galactosidase in E. coli cells.
KW - human interleukin-1β
KW - human leukocyte, fibroblast
KW - optimization of gene expression
KW - overlappons
KW - pig interferons
KW - plasmid vector
KW - Recombinant DNA
KW - translational coupling
UR - http://www.scopus.com/inward/record.url?scp=0025369992&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(90)90069-4
DO - 10.1016/0378-1119(90)90069-4
M3 - Article
C2 - 2187746
AN - SCOPUS:0025369992
VL - 88
SP - 121
EP - 126
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1
ER -
ID: 39444986