Expression and purification of recombinant human MCT-1  oncogene in insect cells.
MCT-1 protein is encoded by an oncogene highly expressed in lymphomas. It is implicated in the interaction with the cap complex of the mRNA, through an RNA binding domain, named PUA. Targeted suppression of this domain attenuates the malignant phenotype and hence MCT-1 is a potential target for therapeutic intervention. In the present study 6 xHis-tagged MCT-1 expression and purification was assessed in insect cells using a baculovirus expression system. The gene was amplified by PCR from a human cDNA library, encoding an open reading frame of 181 amino acid residues. High MCT-1 production level (6 mg/L) was achieved in a two-step purification procedure. The protein was partially characterized by gel filtration chromatography, peptide mass fingerprinting and circular dichroism. A cap-binding assay confirmed its appropriate folding and functionality. Furthermore, a three dimensional model was built based on another known PUA domain structure. The abundant, pure and properly folded source of MCT-1 protein generated lays a foundation for future structure-function studies.
Pérez-Arellano
Isabel
I
Centro de Investigación Príncipe Felipe, Valencia, Spain. iperez@cipf.es
Gozalbo-Rovira
Roberto
R
Martínez
Ana Isabel
AI
Cervera
Javier
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; C115081:MCTS1 protein, human; D015513:Oncogene Proteins; D011994:Recombinant Proteins