Difference between revisions of "Part:BBa K4645000"
(→‎Materials and Method)
 
(16 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K4645000 short</partinfo>
 
<partinfo>BBa_K4645000 short</partinfo>
  
−
Recombinant FelD, produced by <i>E. coli</i>, 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. 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.
+
Recombinant FelD, produced by <i>E. coli</i>, 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 His tag and Flag tag to the end of the C-terminus. 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===
 
===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. 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. 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.  
+
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 d 1 (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 His tag and Flag tag at the C-terminus to better obtain and verify FelD.  
 
===Protein Molecular Structures===
 
===Protein Molecular Structures===
 
<html>
 
<html>
Line 22: Line 22:
 
===Design===
 
===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.  
 
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.  
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
<title>无标题文档</title>
 +
</head>
 +
<body>
 +
<center><img src="https://static.igem.wiki/teams/4645/wiki/feld/feld-design.png" style="width:40%; "></center>
 +
<center><b>Figure 2.</b> Expression construct used for FelD production. </center>
 +
<br>
 +
</body>
 +
</html>
 
===Materials and Method===
 
===Materials and Method===
 
1.Expression and Purification
 
1.Expression and Purification
  
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;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>&nbsp;&nbsp;&nbsp;&nbsp;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>&nbsp;&nbsp;&nbsp;&nbsp;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>&nbsp;&nbsp;&nbsp;&nbsp;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>
Line 33: Line 44:
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Microtiter plates were coated overnight at 4 ℃ with FelD. </p>
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Microtiter plates were coated overnight at 4 ℃ with FelD. </p>
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Plates were blocked with 1% BSA for 1 h at RT. </p>
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Plates were blocked with 1% BSA for 1 h at RT. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Adding 200 ul ELISA Washing Buffer to wash. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Add 200 μl ELISA Washing Buffer to wash. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;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. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) Incubate 100 μl Standard IgE sample (25 ng/μl) or 100 μl sera from allergic donors which was diluted 5-fold at 37℃ for 90 min. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Incubating 100 ul biotin-labeled IgE antibody at 37℃ for 1 h. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Incubate 100 μl biotin-labeled IgE antibody at 37℃ for 1 h. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;6) Adding 350 ul ELISA Washing Buffer to wash 4 time. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;6) Add 350 μl ELISA Washing Buffer to wash 4 time. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;7) Adding 100 ul Streptavidin was labeled by HRP at 37℃ for 30 min. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;7) Add 100 μl HRP-conjugated Streptavidin at 37℃ for 30 min. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;8) Adding 300 ul ELISA Washing Buffer to wash 4 time. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;8) Add 300 μl ELISA Washing Buffer to wash 4 time. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;9) Adding 90 ul color developer (dark) at 37℃ for 15 min. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;9) Add 90 μl color developer (dark) at 37℃ for 15 min. </p>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;10) Adding 50 ul termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;10) Add 50 μl termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength. </p>
  
 
===Result===
 
===Result===
Line 49: Line 60:
 
</head>
 
</head>
 
<body>
 
<body>
−
<center><img src="https://static.igem.wiki/teams/4645/wiki/neua-neub/neua-neub/neua-neub/neua-sds-fig.png" style="width:40%; "></center>
+
<center><img src="https://static.igem.wiki/teams/4645/wiki/feld/feld-purify.png" style="width:40%; "></center>
 
<br>
 
<br>
−
<center><b>Figure 2.</b> SDS-PAGE analysis of FelD with His-tag expression.  </center>
+
<center><b>Figure 3.</b> SDS-PAGE analysis of FelD with His-tag expression.  </center>
 
<br>
 
<br>
  
−
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 2).
+
The molecular weight of FelD is around 18.93 kDa. Thus, the result of SDS-PAGE above indicating that we successfully expressed and purified FelD in the <i>E coli</i> BL21(DE3) <b>(Figure 3)</b>.  
−
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 3).
+
Compared with the control groups, absorbance at 450 nm of IgE from allergic human donor sera has an obvious increase. And we confirmed that FelD can bind to IgE from allergic human donor. The results of our experiment are shown in the figure below <b>(Figure 4)</b>.
 
<html>
 
<html>
 
<head>
 
<head>
Line 62: Line 73:
 
</head>
 
</head>
 
<body>
 
<body>
−
<center><img src="https://static.igem.wiki/teams/4645/wiki/feld/antigen-result.png" style="width:70%; "></center>
+
<center><img src="https://static.igem.wiki/teams/4645/wiki/feld/antigen-result1.png" style="width:70%; "></center>
 
<br>
 
<br>
−
<center><b>Figure 3.</b> OD<sub>450</sub>-Concentration curve of block ability of FelD to IgE from allergic human donor sera or Sstandard IgE sample.  </center>
+
<center><b>Figure 4.</b> OD<sub>450</sub>-Concentration curve of block ability of FelD to IgE from allergic human donor sera or Sstandard IgE sample.  </center>
 
<br>
 
<br>
 
</body>
 
</body>

Latest revision as of 14:05, 12 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 His tag and Flag tag to the end of the C-terminus. 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 d 1 (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 His tag and Flag tag at the C-terminus to better obtain and verify FelD.

Protein Molecular Structures

无标题文档


Figure 1. Protein molecular structures of FelD.

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. 无标题文档

Figure 2. Expression construct used for FelD production.

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) Add 200 μl ELISA Washing Buffer to wash.

    4) Incubate 100 μl Standard IgE sample (25 ng/μl) or 100 μl sera from allergic donors which was diluted 5-fold at 37℃ for 90 min.

    5) Incubate 100 μl biotin-labeled IgE antibody at 37℃ for 1 h.

    6) Add 350 μl ELISA Washing Buffer to wash 4 time.

    7) Add 100 μl HRP-conjugated Streptavidin at 37℃ for 30 min.

    8) Add 300 μl ELISA Washing Buffer to wash 4 time.

    9) Add 90 μl color developer (dark) at 37℃ for 15 min.

    10) Add 50 μl termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength.

Result

无标题文档


Figure 3. SDS-PAGE analysis of FelD with His-tag expression.

The molecular weight of FelD is around 18.93 kDa. Thus, the result of SDS-PAGE above indicating that we successfully expressed and purified FelD in the 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. 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). 无标题文档

Figure 4. OD450-Concentration curve of block ability of FelD to IgE from allergic human donor sera or Sstandard IgE sample.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 467
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]