Assignment task: You have recently discovered a novel protein (Protein X) expressed by human immune cells.
Your initial investigation shows that Protein X has the following characteristics:
- Has 255 amino acid residues and a molecular weight of 35 kDa
- Comprise a single polypeptide chain containing several internal disulphide bonds
- Undergoes posttranslational modifications including N-glycosylation and proteolytic processing
- Is secreted from activated immune cells
- Aids the immune system to better fight infections
You evaluate that there may be a therapeutic/clinical potential of Protein X and therefore set out to produce this glycoprotein with an intension to later administer Protein X to immune compromised patients suffering from immune-deficiencies.
1. Your first strategy is to express Protein X in filamentous fungi i.e. Trichoderma reesei. Answer the following:
a) Why filamentous fungus is a logical first choice
b) The experimental steps (e.g. draw a diagram of an expression cassette and explain the parts)
c) The potential challenges involved with recombinant expression of Protein X in T. reesei
2. To your surprise you are seeing a poor yield of the active form of secreted Protein X from T. reesei.
a) Strategies for expressing Protein X with a reporter molecule that can be used to track the location of the protein with microscopy, flow cytometry and/or other techniques
b) A strategy to improve the yield and/or activity of recombinant Protein X
3. You predict that recombinant Protein X expressed in T. reesei carries N-glycans that differ from the natural glycosylation pattern of human Protein X and that this difference may affect the safety and therapeutic potential of Protein X. Discuss:
a) How glycomics can be used to test if your prediction is correct
b) How the N-glycosylation may impact the function of Protein X
c) A strategy to achieve more human-like glycosylation of recombinant Protein X