Difference between revisions of "Part:BBa K3117006"

Latest revision as of 00:55, 22 October 2019

Igk leader sequence

Usage and Biology

The Igk leader is a signaling sequence, directing the protein into the secretory pathway (Feng et al., 2011). By adding this signaling peptide, the protein of interest can be secreted in the extracellular space after transfection. Prior to secretion the Igk leader is cleaved of.

Characterization

In our project the Igk leader was added to three of our composite parts (BBa_K3117026, BBa_K3117030, BBa_K3117029) expressed in HEK 293T cells for the secretion of our desired proteins into the supernatant.

Figure 1: Western blot of the harvest after transfection of HEK 293T cells with our parts (BBa_K3117026, BBa_K3117030, BBa_K3117029). Anti His-antibody was used for detection

In figure 1 the western blot of the harvested supernatant of HEK 293T cells can be seen. Those proteins were detected via their His-Tag. The presence of the proteins in the supernatant shows that the Igk leader was able to direct all three proteins into the secretory pathway. All proteins had the expected size of around 54 kDa (K1), 42 kDa (K2a), and 41 kDa (K2b). The second band of the K2a transfection sample was unexpected, but does not impact the results in respect to the Igk leaders' successful protein direction into the extracellular space.

By adding this signaling peptide the protein of interest can be secreted in the supernatant after transfection and the basis for a proper protein folding can be provided. Prior to secretion the Igk leader is cleaved of. Igk leader is normally fused to the N-terminus of the protein of interest.

References

Feng, Y., Gong, R., & Dimitrov, D. S. (2011). Design, expression and characterization of a soluble single-chain functional human neonatal Fc receptor. Protein expression and purification, 79(1), 66-71.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3117006 short</partinfo>
 ===Usage and Biology===
-The Igk leader is a signaling sequence, directing the protein into the secretory pathway (Feng et al., 2011). By adding this signaling peptide the protein of interest can be secreted in the supernatant after transfection and the basis for a proper protein folding can provided. Prior to secretion the Igk leader is cleaved of.
+The Igk leader is a signaling sequence, directing the protein into the secretory pathway (Feng et al., 2011). By adding this signaling peptide, the protein of interest can be secreted in the extracellular space after transfection. Prior to secretion the Igk leader is cleaved of.
 ===Characterization===
-In our project the Igk leader was added to three of our composite parts (<partinfo>BBa_K3117026</partinfo>, <partinfo>BBa_K3117030</partinfo>, <partinfo>BBa_K3117029</partinfo>) for the secretion of our desired proteins into the supernatant, after transfection into HEK 293T cells.
+In our project the Igk leader was added to three of our composite parts (<partinfo>BBa_K3117026</partinfo>, <partinfo>BBa_K3117030</partinfo>, <partinfo>BBa_K3117029</partinfo>) expressed in HEK 293T cells for the secretion of our desired proteins into the supernatant.
-[[File:T--FAU_Erlangen--Bild_Sequencing_Ernte_Results_Composite_part.png|thumb|center|200px|'''Figure 2''': Western blot of the harvest after transfection of HEK 293T cells with our parts (<partinfo>BBa_K3117026</partinfo>, <partinfo>BBa_K3117030</partinfo>, <partinfo>BBa_K3117029</partinfo>). Anti His-antibody was used for detection]]
+[[File:T--FAU_Erlangen--Bild_Sequencing_Ernte_Results_Composite_part.png|thumb|center|200px|'''Figure 1''': Western blot of the harvest after transfection of HEK 293T cells with our parts (<partinfo>BBa_K3117026</partinfo>, <partinfo>BBa_K3117030</partinfo>, <partinfo>BBa_K3117029</partinfo>). Anti His-antibody was used for detection]]
-In Fig. 1 the western blot of the harvest of HEK 293T cells can be seen. Those proteins were detected via their His-Tag. The presence of the proteins in the supernatant shows, that the Igk leader was able to direct all three proteins into the secretory way. All proteins had the expected size of around 54 kDa (K1), 42 kDa (K2a), and 41 kDa (K2b). The second band of K2a was unexpected, but does not alter the results that the Igk leader successfully directed our proteins into the extracellular space.
+In figure 1 the western blot of the harvested supernatant of HEK 293T cells can be seen. Those proteins were detected via their His-Tag. The presence of the proteins in the supernatant shows that the Igk leader was able to direct all three proteins into the secretory pathway. All proteins had the expected size of around 54 kDa (K1), 42 kDa (K2a), and 41 kDa (K2b). The second band of the K2a transfection sample was unexpected, but does not impact the results in respect to the Igk leaders' successful protein direction into the extracellular space.
-By adding this signaling peptide the protein of interest can be secreted in the supernatant after transfection and the basis for a proper protein folding can provided. Prior to secretion the Igk leader is cleaved of. Igk leader is normally fused to the N-terminus of the protein of interest.
+By adding this signaling peptide the protein of interest can be secreted in the supernatant after transfection and the basis for a proper protein folding can be provided. Prior to secretion the Igk leader is cleaved of. Igk leader is normally fused to the N-terminus of the protein of interest.
-The Team 2019 FAU_Erlangen used the Igk leader sequence to secrete their proteins (bispecific antibodies) after HEK293T cell transfection.
 ===References===
 Feng, Y., Gong, R., & Dimitrov, D. S. (2011). Design, expression and characterization of a soluble single-chain functional human neonatal Fc receptor. Protein expression and purification, 79(1), 66-71.