Question

Using Protein A as a bait protein in a CO-IP study, protein B and protein C were identified as the binding partners of Protein A in the cell. However, the architectural arrangement of the association of Protein A with protein B and C can be different. As shown in the following diagram, in complex I, both protein B and protein C form direct interactions with Protein A. In complex II, only protein B directly interacts with Protein A, whereas protein C is captured by direct interaction through a common neighbour protein C. Therefore, it is important to examine protein interactions using GST-pull down to check whether the protein-protein interaction is direct or indirect. The information is important to elucidate the function of a specific protein and to develop therapeutics targeting protein-protein interaction.

To examine the internal topology and physical interaction of protein A (~200 amino acids) complex with protein B (~50 amino acids) and protein C (~70 amino acids). Protein B and protein C were expressed and purified as GST-B and GST-C respectively. Protein A was expressed and purified as His6-tagged protein A (His-A). His-tag is about 2 KDa and GST-tag is about 26 KDa.

(2) Two GST-pull down reactions will be performed. GST-pull down assay between His-A and GST-B examines the physical interaction between protein A and protein B, whereas GST-pull down between His-A and GST-C examines the interaction between protein A and protein C. SDS-PAGE will be used to visualize the results of the GST-pull down.

2.1 Predict results of the SDS-PAGE for the protein A:B:C complex as shown in Protein A complex I Input, mix each amount of His-A and GST-B or His-A and GST-C; Elu, the eluted proteins using GST-elution buffer.

Provide a short explanation for your prediction.

2.2 Predict results of the SDS-PAGE for the protein A:B:C complex as shown in Protein A complex II Input, mix each amount of His-A and GST-B or His-A and GST-C; Elu, the eluted proteins using GST-elution buffer.

Provide a brief explanation for your prediction.

Answer 1

Protein A (~200 aa) = 22kDa

Protein B(~50 aa)=5.5kDa

Protein C(~70 aa)=7.7 kDa

2.1 - when you take his-A alone then you observe two bands one at 22kDa and second on 2kDa for his tag.

Gst B alone shows band at 5.5kDa and other band of GST tag on 22kDa.

Input is mainly used as Control HisA and GstB shows bands on 2kDa for HisA and 26kDa of GstB.

In elution His-A and GST B if protein A is interacting with Protein B and Protein C you will get total 5 bands. Three proteins bands for A- 22kDa, B-5.5kDa, C-7.7kDa and other two of tags at 2kDa HisA and 26kDa GstB.

His-A shows same as describe previously.

GST-C alone shows two bands One at 7.7kDa and 26kDa GstC.

Input His-A and GstC shows two bands one of His 2kDa and other for GST at 26kDa.

Elution His-A and GST C shows same bands as Elution His-A and GST B as described previously. This suggest that both Protein B and Protein C interact with Protein A forms complex-1.

2.2- In HisA alone two bands will observe one at 22kDa for protein A and other for His tag at 2kDa.

GSTB alone shows two bands one at 5.5kDa for protein B and other of GST protein at 26kDa.

In input His Aand Gst B two bands will observe one of HisA at 2kDa and other for GST 26kDa.

Elution HisA and GST-B shows 4 bands two for protein A and Protein B at 22kDa and 5.5kDa , other for two tags protein His and Gst at 2kDa and 22kDa.

His A alone same bands as previously describe.

GST-C alone shows two bands one at 7.7kDa for protein C and other for GST at 26kDa.

Input HisA and GST C shows two bands on 2kDa and 26kDa.

Elution His A and GST C shows total four bands two for protein A and Protein C. Other two for tags His and Gst at 2kDa and 26kDa.

Here from Elution HisA and GST-B confirm that their is no interaction observe in protein A or protein B with Protein C. If any interaction of protein observe than Protein C band must be observe in Elution His and GST B.