Expression, purification and characterization of recombinant chicken Thy-1 from Lec-1 mammalian and Tn5 insect cells

Structural studies of glycoproteins are complicated by the inability to generate homogenous samples that readily form ordered crystal. The root cause for this is carbohydrate heterogeneity. Herein we report the cloning, expression, purification, and characterization of a soluble form of avian (chicken) Thy-1. A novel isoform of chicken Thy-1 has been cloned which differs from the previously known chicken isoform by eight amino acid residues. Recombinant protein was expressed in Lec-1 mammalian and Tn5 insect cell-lines, which both produce mannose-rich glycosylations with increased carbohydrate homogeneity compared to the wildtype Chinese Hamster Ovary (CHO) or other mammalian cell lines. The disulfide bond pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT/ICR MS). The glycoforms on Thy-1 from Lec-1 cells were (GlcNAc) ₂(Man)₅ with 20% (GlcNAc)₂(Man)₄, while Thy-1 from Tn5 cells were glycosylated with (GlcNAc)₂(Fuc) _0–2(Man)_2–4. The ability to generate recombinant glycoprotein with increased carbohydrate homogeneity is the first step towards systematic studies of structure-function relationship in intact glycoproteins.