Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, we cloned a fragment of the endogenous murine leukemia viral envelope gene from beta cell line (MIN6N8a) as a new autoantigen gene, whose product was reactive with nonobese diabetic (NOD) mice sera. As a result of determination of nucleotide sequence, this envelope protein had the CKS-17 peptide sequence having immunosuppressive activity. To investigate the role of our cloned transmembrane envelope protein in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM) as an autoantigen or immunosuppressive modulator, a high amount of transmembrane envelope protein was essentially required. Therefore, the expression of our cloned retroviral transmembrane envelope gene was tried in Escherichia coli by a pET vector. However, the expression rate was very low (less than 2%) and most of the expressed protein was insoluble. To improve solubility, the hydrophobic transmembrane anchor domain of our envelope gene was deleted and then the expression of the hydrophilic transmembrane envelope protein was carried out by using the same pET expression system. The expressed protein was completely soluble and the expression yield was dramatically improved by around 25-fold increase. The hydrophilic transmembrane envelope protein was purified by one-step Ni-NTA affinity chromatography and then the fusion tag consisting of the six-histidine peptide and S peptide was removed by cleavage with enterokinase. The processed hydrophilic retroviral transmembrane envelope protein was still immune reactive with NOD sera and also showed immunosuppressive activity by down-regulating the Th1-type cytokine (interferon-gamma) and up-regulating the Th2-type cytokine (interleukin 10).
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PMID:Soluble expression in Escherichia coli of murine endogenous retroviral transmembrane envelope protein having immunosuppressive activity. 1033 56

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr-Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr-Abl proteins, and an intact OD in Bcr-Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr-Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr-Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr-Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni+-NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr-Abl, and potently inhibited the phospho-tyrosine pathways of Bcr-Abl oncoprotein at a low concentration of 4 microM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.
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PMID:Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr-Abl oncoprotein fused to the cytoplasmic transduction peptide. 1904

A novel protocol for producing recombinant Moloney murine leukemia virus (MMLV-RT) in Escherichia coli is reported. The optimized coding sequence for mature MMLV-RT was cloned into pET28a and over-expressed as an N-terminal His6-tagged fusion protein. An enterokinase (EK) recognition site was introduced between the His6-tag and MMLV-RT to release tag-free enzyme. Optimal expression of soluble His6-MMLV-RT was achieved by chaperone co-expression and lower temperature fermentation. The His6-tagged enzyme was first purified by Ni(2+) affinity chromatography. The bound enzyme was then eluted by EK digestion and the eluate was purified on an anion-exchange Q column to remove DNA and EK. Twenty-one milligram MMLV-RT was obtained from 1 l of bacterial culture.
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PMID:A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Escherichia coli. 1933 Apr 87