Expression and codon usage optimization of the erythroid-specific transcription factor cGATA-1 in baculoviral and bacterial systems.
Biochemical characterization of cGATA-1, a key transcription factor in the regulation of globin expression in chickens, has been precluded by the unavailability of appreciable amounts of the pure protein. Purification directly from embryonic red blood cells has been limited by the difficulty in obtaining large quantities of the starting material, and previous attempts at bacterial expression have consistently yielded truncated product. To solve these problems, we have taken two approaches to the expression of cGATA-1. First, we were able to produce efficient expression from baculovirus-infected insect cells. Second, by altering the codon usage in cDNA encoding the protein's carboxy-terminal region, we obtained good expression of full-length protein in Escherichia coli. These preparations should prove useful in biochemical and structural studies of the factor. Additionally, we describe a primer extension/PCR-based method which can be used to synthesize extended regions of DNA sequence for gene construction.
Pikaart
M J
MJ
Laboratory of Molecular Biology, National Institute of Diabetes and Digestion and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
Felsenfeld
G
G
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D003062:Codon; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping; D018411:Spodoptera; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D016335:Zinc Fingers
D003062:Codon; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D015246:Deoxyribonuclease EcoRI