Isopropyl thiogalactoside

Cool the cultures to 30°C and induce protein expression by the addition of isopropyl thiogalactoside (IPTG) to a final concentration of 1 mM and grow for a further 4 hr at 30°C. 

E. coli promoters are those from the lac or trp operons and the late promoter, pL, from phage lambda. These promoters can be turned on by the addition of an inducer (derepressor) such as IPTG (isopropyl thiogalactoside) or lactose for lac, or lAA (indoleacrylic acid) for trp, or by a temperature shift from 30 to 37°C to 42°C for pL. Following process optimization and scale-up, an expression level of 1 to 5 g/liter is achievable with E. coli expression cultures. 

The bacterial lactose-transport protein (lactose permease) transports j3-galactosides, such as lactose, o-nitrophenyl-jS-galactoside, and isopropyl-jS-thiogalactoside. It does not transport galactosides with an a-glycosidic linkage. 

Antibiotics, additives carbenicillin, isopropyl-/3-D-thiogalactoside (IPTG)... 

Induce with isopropyl- S-D-thiogalactoside (IPTG solution) 10 pL of a sterile filtered solution (100 mg mL-1) in pure ethanol. Incubate overnight at 37 °C. 

Transcription ofT7 RNA polymerase is induced by addition of isopropyl-p-D-thiogalactoside. The polymerase then directs the transcription of the lipase operon. The lipase is correctly folded and secreted into the bacterial culture supernatant which can subsequently be used to assay lipase activities. 

The lac operon is ordinarily subject to repression and is activated by the presence of an inducer, now known to be allolactose, o-Galp-pi—>6-d-G1c. However, in experimental work artificial inducers such as isopropyl-p-D-thiogalactoside (IPTG) are most often used. Jacob and Monod postulated that the free repressor protein binds to the operator. In the presence of the inducer a conformational change takes place, destroying the affinity of fhe repressor protein for the operator site. Thus, in the presence of inducer fhe operator is not blocked, and the transcription takes place. Such an operon is said to be negatively controlled and inducible. 

IPTG-Xgal mix Mix 1.25 g IPTG (isopropyl p-D-thiogalactoside) and Xgal (5-bromo-4-chloro-3-indolyi p-D-galactoside) in 25 mL dimethyl formamide (DMF). Store the solution in dark at -20 °C. 

Gilbert and Muller-Hill [215] have isolated a tetra-meric, slightly acidic protein with a molecular weight of 150,000. In vitro studies indicated that the protein has properties compatible with those of a repressor. It combines readily with isopropyl-j -D thiogalactoside (a most powerful inducer of j -galactosidases), as well as with double-stranded DNA. 

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