Expression and characterization of kinase-active v-erbB protein using a baculovirus vector system

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The v-erbB gene, an oncogene from avian erythroblastosis virus, encodes a truncated epidermal growth factor receptor (EGFR).
• Understanding v-erbB protein function and developing methods for its production are important for cancer research.

Purpose of the Study:

• To express functional v-erbB protein in silkworm-derived cells using a Bombyx mori nuclear polyhedrosis virus vector.
• To evaluate the potential for large-scale production of recombinant v-erbB protein.

Main Methods:

• Expression of v-erbB protein and polyhedrin-v-erbB fusion proteins in silkworm cells.
• Utilizing the Bombyx mori nuclear polyhedrosis virus (NPV) vector for protein expression.
• Investigating protein glycosylation using tunicamycin treatment.
• Assessing protein kinase activity through autophosphorylation and substrate phosphorylation assays.

Main Results:

• A polyhedrin-v-erbB fusion protein with a short polyhedrin N-terminus showed over a tenfold increase in expression compared to non-fusion protein.
• Recombinant v-erbB proteins were found to be glycosylated.
• The expressed v-erbB protein demonstrated autophosphorylation of tyrosine residues and phosphorylation of synthetic peptides and lipocortin I, indicating functional kinase activity.

Conclusions:

• Functional v-erbB protein can be successfully expressed in silkworm-derived cells.
• The Bombyx mori NPV system is a viable platform for the large-scale production of recombinant v-erbB protein.
• This system offers a promising approach for generating sufficient quantities of functional v-erbB for further biological and therapeutic studies.