Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system

Area of Science:

• Immunology
• Structural Biology
• Biophysics

Background:

• CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein.
• CD1d binds glycolipid antigens recognized by natural killer T (NKT) cells.
• Understanding CD1d's structural basis for antigen binding and NKT cell recognition is limited.

Purpose of the Study:

• To establish an efficient preparation method for the ectodomain of human and mouse CD1d.
• To improve protein yield and stability for CD1d structural and biophysical studies.
• To evaluate the utility of a silkworm-baculovirus expression system for CD1d production.

Main Methods:

• Utilized a silkworm-baculovirus expression system for producing human and mouse CD1d ectodomains.
• Co-expressed CD1d with β2-microglobulin (β2m).
• Engineered a single-polypeptide construct of CD1d fused with β2m via a flexible linker.
• Employed differential scanning calorimetry (DSC) to assess complex stability and homogeneity.

Main Results:

• Successful co-expression of human and mouse CD1d with β2m, though human CD1d yield was low.
• Engineered single-chained CD1d-β2m complex significantly increased production yield (50 μg/larva).
• The flexible linker enhanced the stability and homogeneity of the CD1d complex, as shown by DSC.

Conclusions:

• The silkworm-baculovirus expression system provides a cost-effective, safe, rapid, and high-yielding method for CD1d production.
• The engineered single-chained construct improves protein yield and complex stability.
• This system is valuable for advancing structural and biophysical investigations of CD1d and NKT cell interactions.