Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When the mammalian aspartic proteinases, procathepsin D or pepsinogen, are expressed in Escherichia coli both accumulate in inclusion bodies. While pepsinogen is efficiently refolded in vitro, recovery of procathepsin D is limited by insolubility. We expressed procathepsin D and pepsinogen in E. coli, with E. coli maltose-binding protein (MBP) or thioredoxin (trx) fused to their C-termini (aspartic proteinase-MBP or aspartic proteinase-trx). The fusion proteins were still found in inclusion bodies. However, the recovery of soluble procathepsin D-MBP and procathepsin D-trx after refolding was facilitated by the bacterial fusion partners. Maltose-binding protein was more efficient than thioredoxin in increasing the recovery of soluble protein. The vector, pET23bMBPH6, can be used for general expression of heterologous proteins in E. coli. The vector includes a histidine tag at the C-terminus of MBP to allow one-step purification of the fusion proteins under denaturing conditions. After purification, the protein of interest can be cleaved from MBP with factor Xa protease and separated from the MBP partner. Refolded pepsinogen-MBP and pepsinogen-trx were enzymatically active, but procathepsin D-MBP and procathepsin D-trx were soluble but largely inactive. The results show that the limited recovery of activity upon refolding of procathepsin D is not the consequence of competing aggregation. Thus, the fusions do not necessarily facilitate native refolding, but they do enhance the recovery of soluble protein. Such fusions could provide a system to study, in soluble form, folding states which are otherwise inaccessible because of aggregation and precipitation.
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PMID:Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin. 947 66

Human pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable. When there were fused to maltose-binding protein (MBP), the fusion proteins (MBP-PGA and MBP-PGC) were expressed as the major products. Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. MBP-PGA and the PGA segment obtained by factor Xa digestion (designated as r-PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t-PGA). For PGCs (MBP-PGC, r-PGC and t-PGC) also, the specific activities were almost the same. However, the activities of PGCs were about 3- to 4-hold higher than those of PGAs. In PGA and PGC immunoassay systems, r-PGs (r-PGA and r-PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation. From these results, r-PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems.
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PMID:Purification of recombinant human pepsinogens and their application as immunoassay standards. 967 50