Expression and Purification of Native Human Granulocyte-Macrophage Colony-Stimulating Factor from anEscherichia coliSecretion Vector
| Autor: | Teresa A. Chiaverotti, Shirley R. Kronheim, Randell T. Libby, David J. Cosman, Gary Braedt, Robert J. Tushinski, Carl J. March, David L. Urdal, Thomas P. Hopp, Diane Y. Mochizuki |
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| Rok vydání: | 1987 |
| Předmět: |
Signal peptide
Recombinant Fusion Proteins Genetic Vectors DNA Recombinant Protein Sorting Signals Lac repressor Biology medicine.disease_cause Biochemistry Colony-Forming Units Assay Bacterial Proteins Colony-Stimulating Factors Complementary DNA Escherichia coli Genetics medicine Humans Secretion Amino Acid Sequence Promoter Regions Genetic Molecular Biology Peptide sequence chemistry.chemical_classification Macrophages DNA Molecular biology Recombinant Proteins Amino acid chemistry Bacterial outer membrane Protein Processing Post-Translational Bacterial Outer Membrane Proteins Granulocytes |
| Zdroj: | DNA. 6:221-229 |
| ISSN: | 0198-0238 |
| Popis: | The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases. |
| Databáze: | OpenAIRE |
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