scholarly journals Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli

1995 ◽  
Vol 311 (3) ◽  
pp. 861-866 ◽  
Author(s):  
R Weinander ◽  
E Mosialou ◽  
J DeJong ◽  
C P D Tu ◽  
J Dypbukt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Expression Vector ◽  
Glutathione Transferase ◽  
Active Form ◽  
Coding Region ◽  
Western Blots ◽  
Cos Cells ◽  
Mammalian Expression ◽  
Microsomal Glutathione

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

scholarly journals Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function

1990 ◽  
Vol 272 (2) ◽  
pp. 479-484 ◽  
Author(s):  
C Andersson ◽  
R Morgenstern
Keyword(s):  
Chemical Modification ◽  
Rat Liver ◽  
Active Site ◽  
Glutathione Transferase ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Histidine Residues ◽  
Catalytic Function ◽  
Low Efficiency ◽  
Microsomal Glutathione

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5′-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

scholarly journals Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues

1991 ◽  
Vol 266 (4) ◽  
pp. 2076-2079
Author(s):  
C Andersson ◽  
E Mosialou ◽  
A E Adang ◽  
G J Mulder ◽  
A van der Gen ◽  
...  
Keyword(s):  
Rat Liver ◽  
Glutathione Transferase ◽  
Microsomal Glutathione

Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli

1989 ◽  
Vol 3 (1) ◽  
pp. 15-21 ◽  
Author(s):  
M. C. Hanks ◽  
R. T. Talbot ◽  
H. M. Sang
Keyword(s):  
Escherichia Coli ◽  
Cdna Sequence ◽  
Expression Vector ◽  
Total Cell ◽  
Biologically Active ◽  
Cell Protein ◽  
Western Blots ◽  
E Coli ◽  
Ring Dove

ABSTRACT The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK233-2 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1·5% of total cell protein.

Antibodies induced in mice by a DNA-construct coding for the elastase of Schistosoma mansoni recognize the enzyme in secretions and preacetabular glands of cercariae

Parasitology ◽  
2002 ◽  
Vol 124 (3) ◽  
pp. 301-306 ◽  
Author(s):  
M. BAHGAT ◽  
K. CHLICHLIA ◽  
V. SCHIRRMACHER ◽  
A. RUPPEL
Keyword(s):  
Schistosoma Mansoni ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Transcriptional Level ◽  
Western Blots ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Gene Coding ◽  
Native Antigen ◽  
First Time

A DNA-construct coding for the elastase of the parasite Schistosoma mansoni was prepared from adult S. mansoni worm RNA which was reverse transcribed into cDNA. The gene coding for the elastase was amplified using primers specific for the sequence of cercarial elastase and was cloned into a mammalian expression vector. Expression of the elastase gene at the transcriptional level was achieved for the first time in transfected mammalian cells (COS-7) and was also successful in muscle tissue of mice injected with the DNA-construct. These mice developed antibodies recognizing in Western blots the elastase from cercarial secretions. Also, these antibodies reacted in immunofluorescence tests with the preacetabular glands of cercariae, i.e. the site of origin for elastase. Thus, the DNA-construct induced the expression of elastase in mice and formation of antibodies that recognized the native antigen.

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