PMID: PMC4084840-1-1

 

    Legend: Gene, Sites

Title : Purification of ITIH4 fromCell Media and Human Serum

Abstract :

1. Recombinant ITIH4 was isolatedfrom the cell media of HEK293 cells overexpressing ITIH4-cMyc-DDKprotein by ultrafiltration followed by RP-1S chromatography
2. Afterthese steps, ITIH4 exceeded 90% purity based on SDS-PAGE with Coommassieblue staining (data not shown)
3. Proteomic analysis of a tryptic digestconfirmed the identity of ITIH4 (71% sequence coverage) with minimalprotein impurities
4. To purify ITIH4 from serum, we devised a three-steppurification process to minimize biased selection of specific glycoformsby the chromatographic steps (Figure 1A)
5. Ammoniumsulfate precipitation resulted in a 5-fold enrichment of ITIH4 witha yield of 84% (Table 1)
6. Further enrichmentwas achieved by Cibacron Blue chromatography (3-fold enrichment) followedby reversed-phase chromatography on a monolithic protein purificationcolumn (10-fold enrichment)
7. The observed mass shift of recombinantand serum ITIH4 on SDS-PAGE after treatment with PNGaseF confirmedthat ITIH4 was heavily N-glycosylated (Figure 1B)

Output (sent_index, trigger, protein, sugar, site):

Output(Part-Of) (sent_index, protein, site):

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX