Gene cloning, expression, and characterization of the Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius.
The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced. The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively. Southern blots indicate that these are the only two Sac7 protein genes in S. acidocaldarius, each present as a single copy. Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species. The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements. Potential ribosome binding sites have been identified downstream of the initiator codons. The sac7d gene has been expressed in E. coli, and various physical properties of the recombinant protein have been compared with those of native Sac7. The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds. The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly[dGdC].poly[dGdC] in 0.01 M KH2PO4 at pH 7.0. The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein. Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C.
McAfee
J G
JG
Department of Medical Biochemistry, School of Medicine, Southern Illinois University, Carbondale 62901-4413, USA.
Edmondson
S P
SP
Datta
P K
PK
Shriver
J W
JW
Gupta
R
R
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D005798:Genes, Bacterial / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D016945:Sulfolobus acidocaldarius / Q000235:genetics*; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C093228:Sso7d protein, Sulfolobus; C095116:Sac7 protein, Sulfolobus