Production of soluble hDll1^NDSL and GST-hDll1^NDSL recombinant proteins.

(A) Amino acid sequences of hDll1 and mDll1 containing the N-terminal and DSL regions are termed hDll1^NDSL and mDll1^NDSL. The two sequences are highly conserved with 92% identity and 97% similarity in amino acid sequences. The cDNA sequences encoding hDll1^NDSL was subcloned into pGEX-2T vector to create GST-hDll1^NDSL fusion protein. (B) Production of hDll1^NDSL by digestion of the fusion protein with thrombin protease. Coomassie-blue staining of the protein gel is shown. Five microliters DEAE fraction, DEAE column pass and wash (fractions 1 and 2) before digestion, and column pass and elute after digestion were analyzed by SDS-PAGE. 1, indicates the position of GST-hDll1^NDSL at 43-kd marker; 2, indicates GST at 26 kd; 3, indicates hDll1^NDSL at 17 kd. (C). Western blot analysis of the recombinant proteins. Monomeric forms of thioredoxin-mDll1^NDSL, GST-hDll1^NDSL, and hDll1^NDSL were detected at positions 34 kd, 43 kd, and 17 kd, respectively by Western blot with anti-mDll1^NDSLantibody. Polymers of the two fusion proteins were also revealed. No staining for GST was seen. A weak band in the hDll1-NDSL lane at approximately 27 kd was detected. It is highly possible that the band represented a degraded or partially digested product of GST-hDll1-NDSL during overnight incubation with thrombin protease.