EGF Transportation

From 2009.igem.org

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Latest revision as of 22:36, 21 October 2009

EGF-TliA, EGF-LARD1 fusion proteins were secreted when ABC transporter was present. E. coli harboring the PrtDEF of E. chrysanthemi secreted more fusion proteins than without ABC transporter. In fact, in enzyme activation assay(with Hitachi Spectrophotometer) we observed that although our protein -EGF- has disulfide bonds, it was excreted succesfully. In addition, when compearing with lack of ABC domain, we saw that protein excretion into medium (supernatant) is 10-fold more with ABC transporter-PrtDEF-. PrtDEF functioned well at 37°C, the optimum growth temperature for E. coli. In our project Wound Dressing, for excretion of EGF and KGF into supernatant,we used the plasmid.It is a device which contains three genes-prtD,prtE and prtF- in order to form ATP Binding Cassette(ABC) transporter domain on E.coli membrane.Thus,the plasmid is not used for digestion or ligation.The plasmid works with constutively on "tetR" promoter.

[1]PrtDEF: ABC transporter of Erwinia chrysanthemi for PrtA
[2]EGF: Human Epidermal Growth Factor

Figure:Proposed model to explain blockage of secretion for proteins containing disulfide bonds. (A) The hybrid carrying a preformed disulfide bond approaches the channel entrance, is recognized by the secretion machinery (PrtD), but cannot go across the channel. (B) Proteins are stacked inside the channel during secretion. Disulfide bonds may be formed at this stage. (C)Inclusion body formations of protein can not enter into the domain nor pass through the domain.

Whole plasmid ,working contituvely on, is used and not required to be digested or ligated, since 3 genes involve in order to form ABC transporter domain. We used the plasmid with our signal tags LARD1 and TliA.

To go LARD1 :

To go TliA :

Experiments with TliA

Tributyrin : It is a triglyceride naturally present in butter. It is an ester composed of butyric acid and glycerol.

Chemical Formula: C15-H26-O6
Molecular Weight: 302.37 g/mole

Lipase : It is a soluble enzyme that catalyzes the hydrolysis of ester bonds in water–insoluble, lipid substrates.

ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) is secreted through the ABC transporter. TliA has four glycine rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. Whole TliA is the longest LARD. Lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins. Result of our experiment There was TliA-AbC in the LB Agar+Chm+Amp+Try plate so lipase reacted with tributyrin and butyric acid was formed. As a result of this, we have seen the zone formation.

We holed 3mm zone in the plate and we poured the supernatant. 2 of them was including E.coli with ABC-transporter and 2 of them did not contain. After that we left these plates in incubation at 25 C for 2 days.

As we expected, area of zone in plate with ABC transporter is larger than the without ABC transporter.

Enzyme Kinetic Assay Lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (pNPP) as a substrate. Ten millimolar pNPP dissolved in acetonitrile was mixed with ethanol and 50 mM Tris-HCl (pH 8.5) to a final ratio of acetonitrile:ethanol:Tris-HCl of 1:4:95 (v/v/v). The reaction was started by adding 50 μl of culture supernatant to 200 μl of reaction mixture at 42°C, and absorbance at 420 nm was monitored with a Hitachi spectrophotometer (Enzyme Kinetic Activity Assay)

Applications of BBa_K258006

While the secretory phenotype of fusion proteins with TliA was evident based on lipase activity, secretion of fusion proteins with LARDs could be detected by Western blotting.

Lipase activity To identify a secretion phenotype on solid medium, E. coli was grown at 25°C for 48 h on LAT (LB medium, 1.5% Bacto Agar, 0.5% tributylin). The phenotype was evident by the development of a halo due to the secreted lipase [12]. In addition, lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (pNPP) as a substrate [12]. Ten millimolar pNPP dissolved in acetonitrile was mixed with ethanol and 50 mM Tris-HCl (pH 8.5) to a final ratio of acetonitrile:ethanol:Tris-HCl of 1:4:95 (v/v/v). The reaction was started by adding 50 μl of culture supernatant to 200 μl of reaction mixture at 42°C, and absorbance at 420 nm was monitored with Hitachi Spectrophotometry for 20 min. The activity was measured by the increase of optical density (OD).

procedure from:

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD) Chan Woo Chung, Jinsun You, Kyeongyeon Kim, Yuseok Moon, Hoeon Kim and Jung Hoon Ahn* Published: 29 January 2009 Microbial Cell Factories 2009, 8:11 doi:10.1186/1475-2859-8-11 Received: 18 November 2008 Accepted: 29 January 2009 This article is available from: http://www.microbialcellfactories.com/content/8/1/11