Protocol for Assay

Draft v 1

Experimental Protocol: CBM27_RGD_MaSp1_4x Fusion Expression and Tremella Composite Formation

Construct: CBM27_RGD_MaSp1_4x_Fusion Vector: pET28a Expression system: NEB PURExpress E6800 Protein MW: ~32 kDa

Step 1: Cell-Free Expression

Materials needed:

• PURExpress Solution A
• PURExpress Solution B
• Murine RNase Inhibitor
• Template DNA (linear PCR product or circular plasmid, 250 ng)
• Nuclease-free water
• Ice

Protocol:

1. Thaw Solutions A and B on ice. Do not vortex.
2. Assemble the following reaction on ice in this exact order in a 1.5 ml microcentrifuge tube:
   • 10 µl Solution A
   • 7.5 µl Solution B
   • 0.5 µl RNase Inhibitor (20 units)
   • 2 µl template DNA (250 ng)
   • 5 µl nuclease-free water
   • Total volume: 25 µl
3. Mix gently by pipetting up and down 5 times. Do not vortex.
4. Incubate at 37°C for 2 hours.
5. Place on ice immediately after incubation.

Run alongside: one negative control reaction with no template DNA.

Step 2: SDS-PAGE Confirmation

Materials needed:

• 10-20% Tris-glycine precast gel
• SDS loading buffer
• Protein ladder (10-250 kDa range)
• Running buffer
• Coomassie Blue stain

Protocol:

1. Take 2.5 µl of the expression reaction and add 2.5 µl SDS loading buffer.
2. Heat at 95°C for 5 minutes to denature proteins.
3. Load onto gel alongside protein ladder and negative control.
4. Run at 200V for 35 minutes.
5. Stain with Coomassie Blue for 1 hour, destain with water overnight.
6. Look for a band at approximately 32 kDa.

Success criterion: A visible band at 32 kDa in the expression lane that is absent in the negative control lane.

Step 3: Tremella Composite Formation (only if Step 2 successful)

Materials needed:

• Tremella fuciformis dried polysaccharide (commercially available, e.g. Sigma or specialist supplier)
• PBS buffer pH 7.4
• Your expressed fusion protein from Step 1 (remaining ~22.5 µl)
• Microcentrifuge tubes
• Rotary mixer or gentle agitation

Protocol:

1. Prepare a 1% w/v Tremella polysaccharide solution by dissolving 10 mg dried Tremella polysaccharide in 1 ml PBS pH 7.4. Stir gently at room temperature for 2 hours until fully dissolved. This produces a viscous gel solution.
2. Take the remaining expressed fusion protein from Step 1 (~22.5 µl).
3. Add 22.5 µl of the 1% Tremella polysaccharide solution to the protein.
4. Mix gently by pipetting 10 times. Do not vortex.
5. Incubate at room temperature for 30 minutes on a rotary mixer at low speed to allow CBM27 domain binding to the Tremella mannan backbone.
6. Observe for gelation. The mixture should become more viscous than either component alone if the CBM27 is anchoring the silk to the Tremella matrix.

Success criterion: Visible increase in viscosity or gel formation compared to a control mixture of the same volume of PURExpress reaction without the fusion protein mixed with Tremella.

What Each Result Means