Purification and characterization of a recombinant rat prohaptoglobin expressed in baculovirus-infected Sf9 insect cells
| Autor: | Gail Otulakowski, Alexander Hillar, Hugh O′Brodovich |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
DNA
Complementary viruses Enzyme-Linked Immunosorbent Assay Sf9 Spodoptera Biology Recombinant virus Polymerase Chain Reaction law.invention Shuttle vector law Complementary DNA Animals Protein Precursors DNA Primers Expression vector Base Sequence Haptoglobins Hydrolysis fungi Nuclear Polyhedrosis Virus biology.organism_classification Molecular biology Recombinant Proteins Rats Autographa californica Biochemistry Recombinant DNA Protein Processing Post-Translational Biotechnology |
| Zdroj: | Protein Expression and Purification. 55:246-256 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2007.06.004 |
| Popis: | To generate hemoglobin-free full-length haptoglobin the cDNA encoding rat haptoglobin alphabeta subunits was cloned into shuttle vector pVT-Bac-His and used to produce a recombinant baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) as an expression vector, named HpAcNPV. Recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The 50 kDa protein expressed was mostly secreted into the culture medium at relatively high titer (15 microg/mL) and was found to be rat prohaptoglobin having a vector-derived N-terminal extension of 37 amino acids, containing both a hexahistidine tag and an enterokinase recognition sequence. The protein was successfully purified by a three step procedure including nickel-linked agarose and DEAE-Sepharose chromatography steps. Hemoglobin was not detected in the purified preparations. Purified recombinant rat prohaptoglobin protein was also found to be glycosylated, and to be capable of forming a complex with rat hemoglobin in vitro. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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