This Mouse P4HB overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of P4HB protein (Cat: 50638-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse P4HB (NP_035162.1) (Met 1-Lys 506) was expressed, with a C-terminal polyhistidine tag.
The secreted recombinant mouse P4HB comprises 498 amino acids and has a calculated molecular mass of 56.2 kDa as estimated in SDS-PAGE under reducing conditions.
Mouse P4HB HEK293 Overexpression Lysate: 使用指南
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Protein disulfide-isomerase, also known as Cellular thyroid hormone-binding protein, Prolyl 4-hydroxylase subunit beta, p55 and P4HB, is a peripheral membrane protein that belongs to the protein disulfide isomerase family. P4HB is highly abundant. In some cell types, it seems to be also secreted or associated with the plasma membrane, where it undergoes constant shedding and replacement from intracellular sources. P4HB localizes near CD4-enriched regions on lymphoid cell surfaces. It is identified by mass spectrometry in melanosome fractions from stage I to stage IV. P4HB reduces and may activate fusogenic properties of HIV-1 gp12 surface protein, thereby enabling HIV-1 entry into the cell. P4HB catalyzes the formation, breakage and rearrangement of disulfide bonds. At the cell surface, it seems to act as a reductase that cleaves disulfide bonds of proteins attached to the cell. P4HB may therefore cause structural modifications of exofacial proteins. Inside the cell, it seems to form/rearrange disulfide bonds of nascent proteins. At high concentrations, P4HB functions as a chaperone that inhibits aggregation of misfolded proteins. At low concentrations, it facilitates aggregation (anti-chaperone activity). P4HB may be involved with other chaperones in the structural modification of the TG precursor in hormone biogenesis. It also acts as a structural subunit of various enzymes such as prolyl 4-hydroxylase and microsomal triacylglycerol transfer protein MTTP.
prolyl 4-hydroxylase, beta polypeptide
Kivirikko KI, et al., 1989, FASEB J., 3 (5): 1609-17.
Pihlajaniemi T, et al.,1991, J Hepatol., 13, Suppl 3: S2
Fenouillet E., et al., 2001, J. Infect. Dis. 183:744-752.
Gevaert K., et al., 2003, Nat. Biotechnol. 21:566-569.
Barbouche R., et al., 2003, J. Biol. Chem. 278:3131-3136.