v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl beta-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.
Bacterial Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Glutathione Transferase ; biosynthesis ; genetics ; isolation & purification ; Oncogene Protein pp60(v-src) ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; isolation & purification ; Saccharomyces cerevisiae ; genetics ; metabolism

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Adaptor Proteins, Signal Transducing ; Carrier Proteins ; chemistry ; Chemical Precipitation ; DNA-Binding Proteins ; chemistry ; GATA1 Transcription Factor ; chemistry ; Genetic Vectors ; Glutathione Transferase ; chemistry ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; chemistry ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Renaturation ; Proto-Oncogene Proteins ; chemistry ; Recombinant Fusion Proteins ; genetics ; metabolism

A fusion gene between echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) has been identified in non-small cell lung cancers (NSCLCs). Although a few studies have evaluated EML4-ALK fusion genes in Korean NSCLCs, the prevalence of different EML4-ALK fusion variants has yet to be clearly assessed. Herein, we have examined the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLCs. EML4-ALK fusion genes have been detected in 10 (6.0%) of 167 patients of NSCLCs and in 9 (7.4%) of 121 patients of adenocarcinoma. Of the 10 patients with fusion genes identified, 8 (80%) were E13;A20 (variant 1) and 2 (20%) were E6;A20, with an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). These results indicate that the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLC may differ from those in other ethnic populations. Herein, we describe for the first time the profiles of EML4-ALK fusion variants of Korean patients with NSCLCs.
Adenocarcinoma/diagnosis/genetics ; Aged ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Carcinoma, Non-Small-Cell Lung/diagnosis/*genetics ; Exons ; Female ; Humans ; Introns ; Lung Neoplasms/diagnosis/*genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion/chemistry/*genetics ; Republic of Korea ; Sequence Analysis, RNA ; Smoking

OBJECTIVETo determine the ETO-interaction domain of nuclear receptor co-repressor (N-CoR) for abolishing the biological function of AML1-ETO.

METHODSTen different regions of N-CoR (N-CoRYs) were generated by means of polymerase chain reaction (PCR), and cloned into yeast expression plasmid pGADGL to construct pGADGL/N-CoRYs. The yeast two-hybrid technique and X-gal staining were used to determine the binding between the 10 different regions of N-CoR and ETO.

RESULTSIt was shown that the co-existence of 988-1,126 and 1,551-1,803 amino acid residues of N-CoRY was the ETO-interaction domains required for the binding with ETO.

CONCLUSIONTwo domains of N-CoR that interact with two zinc fingers of ETO, and keep stable binding between the two proteins were identified.

Binding Sites ; genetics ; Humans ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Nuclear Receptor Co-Repressor 1 ; Plasmids ; genetics ; Protein Binding ; Proto-Oncogene Proteins ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Recombinant Fusion Proteins ; genetics ; metabolism ; Repressor Proteins ; chemistry ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques

The study was purposed to analyze the relationship between the content of T-cell receptor excision DNA circles (TREC) and bcr-abl mRNA levels in CML patients and to evaluate the prognostic significance of recent thymic output function detection in patients with chronic myelogenous leukemia (CML). Quantitative detection of TREC and bcr-abl fusion gene transcripts in peripheral blood from 15 CML patients were preformed by real-time PCR. The change of bcr-abl levels in 6 patients was followed-up for two years. The results showed that there was no significant correlation between TREC and bcr-abl mRNA levels in peripheral blood from CML patients for the first attack. Patients who had higher TREC at diagnosis had a larger reduction of bcr-abl after 2 years of follow-up. While out of 2 patients who underwent haemopoietic stem cell transplantation (HSCT), one patient with higher level of TREC before transplantation was confirmed to express undetectable level of TREC by three consecutive detections after transplantation, other one patient was identified to express low level of bcr-abl. It is concluded that high thymic output function in CML patients can be beneficial for killing the residual CML cells.
Adolescent ; Adult ; Aged ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Gene Rearrangement ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-abl ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcr ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Antigen, T-Cell ; analysis ; immunology ; T-Lymphocytes ; chemistry ; immunology ; Thymus Gland ; immunology

Promyelocytic leukemia (PML) protein, a tumor suppressor, plays an important role in patients with acute promyelocytic leukemia (APL) receiving arsenic trioxide (AsO) therapy. APL is a M3 subtype of acute myeloid leukemia (AML), which is characterized by expression of PML-RARα (P/R) fusion protein, leading to the oncogenesis. AsO is currently used as the first-line drug for patients with APL, and the mechanism may be:AsO directly binds to PML part of P/R protein and induces multimerization of related proteins, which further recruits different functional proteins to reform PML nuclear bodies (PML-NBs), and finally it degraded by SUMOylation and ubiquitination proteasomal pathway. Gene mutations may lead to relapse and drug resistance after AsO treatment. In this review, we discuss the structure and function of PML proteins; the pathogenesis of APL induced by P/R fusion protein; the involvement of PML protein in treatment of APL patient with AsO; and explain how PML protein mutations could cause resistance to AsO therapy.
Antineoplastic Agents ; therapeutic use ; Arsenic Trioxide ; therapeutic use ; Drug Resistance, Neoplasm ; genetics ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; Mutation ; Oncogene Proteins, Fusion ; metabolism ; Promyelocytic Leukemia Protein ; chemistry ; genetics ; metabolism

Base Sequence ; Formaldehyde ; chemistry ; Humans ; Molecular Sequence Data ; Oncogene Proteins, Fusion ; genetics ; Paraffin Embedding ; Proto-Oncogene Protein c-fli-1 ; genetics ; RNA, Neoplasm ; genetics ; metabolism ; RNA-Binding Protein EWS ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rhabdomyosarcoma ; genetics ; Sarcoma, Ewing ; genetics ; Sarcoma, Synovial ; genetics ; Soft Tissue Neoplasms ; genetics ; Tissue Fixation

OBJECTIVETo further verify the transcriptional repression domains in ETO and their relationship with histone deacetylase (HDAC).

METHODSEither of the ETO two zinc fingers was mutated respectively by site-directing mutagenesis. The truncation fragments of ETO were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic expression plasmid pFA-CMV. By the means of DNA transfection and analysis of the transcription derived from the promoter of reporter gene, the transcriptional regulation domains of ETO was determined.

RESULTSThe expression plasmids carrying truncated ETO and ETO with point mutation at either zinc finger were successfully constructed. Two repression domains were found within ETO, which were located at two zinc finger motifs and 275 - 487 amino acid residues, respectively.

CONCLUSIONThe transcription repression by ETO was mediated by two separated domains and closely associated with HDAC, which may be used as therapeutic target for acute myeloid leukemia M(2b).

Core Binding Factor Alpha 2 Subunit ; chemistry ; genetics ; Gene Expression Regulation, Leukemic ; genetics ; Genetic Vectors ; Histone Deacetylases ; physiology ; Humans ; In Vitro Techniques ; Leukemia, Myeloid, Acute ; enzymology ; genetics ; Oncogene Proteins, Fusion ; chemistry ; genetics ; RUNX1 Translocation Partner 1 Protein ; Transcription, Genetic ; Transfection ; Zinc Fingers ; genetics

Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3's function as an allosteric activator and its role in downstream signaling.
Amino Acid Sequence ; Animals ; CHO Cells ; Cell Movement ; Cell Proliferation ; Cricetinae ; Cricetulus ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Intracellular Space ; enzymology ; MAP Kinase Signaling System ; Models, Molecular ; Molecular Sequence Data ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptor, ErbB-2 ; chemistry ; Receptor, ErbB-3 ; chemistry ; genetics ; metabolism ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; Signal Transduction