Difference between revisions of "Part:BBa K4645000"
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1.Expression and Purification | 1.Expression and Purification | ||
| − | <p> 1) Plasmid pET-28a(+)-FelD (with His-tag) is transformed to <i>Escherichia coli</i> BL21 (DE3). The E. coli strain is cultured in LB medium containing 50 μg/mL kanamycin. </p> | + | <p> 1) Plasmid pET-28a(+)-FelD (with His-tag) is transformed to <i>Escherichia coli</i> BL21(DE3). The E. coli strain is cultured in LB medium containing 50 μg/mL kanamycin. </p> |
<p> 2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 2 mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). </p> | <p> 2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 2 mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). </p> | ||
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| − | The molecular weight of FelD is around 18.93 kDa. Thus, the result of SDS-PAGE above indicating that there do have the expression of FelD in our chassis <i>E. coli</i> BL21 (DE3) (Figure 3). | + | The molecular weight of FelD is around 18.93 kDa. Thus, the result of SDS-PAGE above indicating that there do have the expression of FelD in our chassis <i>E. coli</i> BL21(DE3) (Figure 3). |
Compared with the control groups, absorbance at 450 nm of IgE from allergic human donor sera has an obvious increase. We successfully express and purify the FelD in the Escherichia coli. And we confirmed that FelD can bind to IgE from allergic human donor. The results of our experiment are shown in the figure below (Figure 4). | Compared with the control groups, absorbance at 450 nm of IgE from allergic human donor sera has an obvious increase. We successfully express and purify the FelD in the Escherichia coli. And we confirmed that FelD can bind to IgE from allergic human donor. The results of our experiment are shown in the figure below (Figure 4). | ||
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Revision as of 06:09, 8 October 2023
FelD: Felis domesticus allergen
Recombinant FelD, produced by E. coli, exhibits comparable immunogenicity to Fel d 1 extracted from cats and is capable of inducing allergies in the human body. Its structure comprises the 93 amino acids of chain 2 of natural FelD that is directly fused to the 70 amino acids of chain 1 [1]. This is followed by the addition of 6*His tag and Flag tag to the end of the C-terminal. By binding with IgE in individuals allergic to cats, FelD mediates allergy symptoms in the affected individuals. In our study, FelD was utilized to investigate the blocking effect of our scFvs (single-chain fragment variables) on cat allergies.
Usage and Biology
Of all known cat allergens, feline domestic allergen 1 (Fel d 1) is the most well-studied. IgE antibodies to Fel d 1 are present in sera in more than 90%±95% of feline allergy patients. Fel d 1 is a 38 kDa acidic glycoprotein with N-linked carbohydrate content, found in feline pelt, saliva and lacrimal glands. The allergen consists of two 18 kDa non-covalently linked heterodimers,, each consisting of two chains, chain 1 (8 kDa) and chain 2 (10 kDa), encoded by different genes [2]. Using the direct fusion of 93 amino acids of chain 2 and 70 amino acids of chain 1, we succeeded in creating in vitro conditions for proper folding of both chains. Stable recombinant Fel d1 (FelD) acts in a very similar way to natural allergens. It displays the same disulfide bond pattern as the natural protein and forms homodimers with a similar secondary structure [3]. Most importantly, it also acts as a native allergen in terms of in vitro immune reactivity. In addition, we combined 6*His tag and Flag tag at the C-terminal to better obtain and verify FelD.
Protein Molecular Structures
Design
The plasmid we designed consists of T7 promoter, lac operator, RBS, FelD coding sequence, His tag, Flag tag and T7 terminator, which are arranged in an order on a pET28 backbone. We aim to induce the transcription of the downstream FelD by adding the IPTG to initiate the expression. The protein will then be purified and block activity to IgE was tested by Elisa. We determine the binding by coating FelD in the microtiter plates and by measuring the absorbance.
Materials and Method
1.Expression and Purification
1) Plasmid pET-28a(+)-FelD (with His-tag) is transformed to Escherichia coli BL21(DE3). The E. coli strain is cultured in LB medium containing 50 μg/mL kanamycin.
2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 2 mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China).
2.Elisa
1) Microtiter plates were coated overnight at 4 ℃ with FelD.
2) Plates were blocked with 1% BSA for 1 h at RT.
3) Adding 200 ul ELISA Washing Buffer to wash.
4) Incubating 100 ul Standard IgE sample (25 ng/ul) or 100 ul sera from allergic donors which was diluted 5-fold at 37℃ for 90 min.
5) Incubating 100 ul biotin-labeled IgE antibody at 37℃ for 1 h.
6) Adding 350 ul ELISA Washing Buffer to wash 4 time.
7) Adding 100 ul Streptavidin was labeled by HRP at 37℃ for 30 min.
8) Adding 300 ul ELISA Washing Buffer to wash 4 time.
9) Adding 90 ul color developer (dark) at 37℃ for 15 min.
10) Adding 50 ul termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength.
Result
The molecular weight of FelD is around 18.93 kDa. Thus, the result of SDS-PAGE above indicating that there do have the expression of FelD in our chassis E. coli BL21(DE3) (Figure 3). Compared with the control groups, absorbance at 450 nm of IgE from allergic human donor sera has an obvious increase. We successfully express and purify the FelD in the Escherichia coli. And we confirmed that FelD can bind to IgE from allergic human donor. The results of our experiment are shown in the figure below (Figure 4).
Reference
[1]Kaiser L, Grönlund H, Sandalova T, Ljunggren HG, van Hage-Hamsten M, Achour A, Schneider G. The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family. J Biol Chem. 2003 Sep 26;278(39):37730-5.
[2] Rogers BL, Morgenstern JP, Garman RD, Bond JF, Kuo MC. Recombinant Fel d.I: Expression, purification, IgE binding and reaction with cat-allergic human T cells. Molecular Immunology. 1993 Apr;30(6):559-568.
[3] Kaiser L, Grönlund H, Sandalova T, Ljunggren HG, Schneider G, van Hage-Hamsten M, Achour A. Production, crystallization and preliminary crystallographic study of the major cat allergen Fel d 1. Acta Crystallogr D Biol Crystallogr. 2003 Jun;59(Pt 6):1103-5.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 467
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]