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Cytochrome P450scc (P450scc) catalyzes the first step in steroid hormone synthesis, the conversion of cholesterol to pregnenolone.
The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin.
The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach.
2.2.1. Starting plasmids
The vector for expression of bovine AdR, plasmid pBAR1607 (Sagara et al., 1993), was kindly provided by Yasuhiro Sagara. This vector is a derivative of pCWori+, containing a AdR cDNA fragment inserted between the NdeI and KpnI sites of the polylinker.
The plasmids pKKHC-Adx (Uhlmann et al., 1992) and pKKHC-CYP106A2 (Simgen et al., 2000) provided the templates for PCR amplification of Adx and CYP106A2, respectively.
Expression vector pTrc99a (Amann et al., 1988) and cloning vector pACYC184
(Chang and Cohen, 1978) were used for constructing the new expression vector for CYP106A2.
The vector for expression of bovine AdR, plasmid pBAR1607 (Sagara et al., 1993), was kindly
provided by Yasuhiro Sagara. This vector is a derivative of pCWori+, containing a AdR cDNA fragment inserted between the NdeI and KpnI sites of the polylinker. The plasmids pKKHC-Adx (Uhlmann et al., 1992) and pKKHC-CYP106A2 (Simgen et al., 2000) provided the templates for PCR amplification of and CYP106A2, respectively.
2.2.2. Construction of the plasmid pBAR Twin
Oligonucleotides encoding the amino- and carboxytermini of the truncated bovine Adx4-108 were used as primers for amplification of the cDNA fragment from plasmid pKKHC-Adx. The forward primer was designed with a restriction site (KpnI) immediately upstream of the ribosome binding site and the coding region and the reverse primer contains a stop codon followed by an EcoRI restriction site to facilitate cloning into the expression vector pBAR1607.
The 0.4-kb fragment was ligated between the KpnI and EcoRI sites of pBAR1607. In the resulting construct pBAR Twin the cDNAs of AdR and Adx orientated in tandem are under control of a lac and two tac promoters (Fig. 2).
Fig. 2. CYP106A2 and AdR/Adx coexpression plasmids. The 6.7-kb vector pBAR Twin contains two copies of the tac and one lacUV5 promoter, the cDNAs from AdR and Adx, the _-lactamase gene, and the high-copy origin of replication of the pUC series of plasmids. CYP106A2 is encoded on the 5.4 kb construct pACYC FHH2 8 under control of a tac promoter and a transcription terminator derived from the rrnB gene. The plasmid features the low-copy pMB1 origin of replication and the sequence of the cat resistance gene.
Cytochrome P450scc (P450scc)3 catalyzes the three hydroxylation reactions required to convert cholesterol to pregnenolone, the first step in steroid hormone biosynthesis.
Electrons for the hydroxylation reactions are supplied by NADPH via the electron transfer proteins adrenodoxin reductase and adrenodoxin.
Bovine and human cytochrome P450scc genes (CYP11A1) encode 520 and 521 amino acid proteins, respectively.
Bacterial expression of P450scc.
The human placental cDNA library was from Clontech.
Initially the library was screened with antiserum to bovine P450scc (15–17). The resulting 50 plaques producing a positive signal were screened by hybridization using the synthetic oligonucleotide 59-ATGTTCCACACCAGCCTCCCCATG-39, end labeled with [32P]phosphate (17). From this a full-length cDNA was isolated. PCR was used to remove the precursor sequence from the P450scc cDNA to enable bacterial expression of the mature protein. A synthetic oligonucleotide containing EcoRI and NcoI sites and the initiator codon ATG within the NcoI site (underlined) was used as a 59-primer and an oligonucleotide containing a KpnI site (underlined) was used as the 39-primer. The sequences of the primers were as follows:
5’ primer, 5’-GGGAATTCCATGGCAAGTACAAGAAGTCCTCGCCCCTTCAATGAG-3’ 3’ primer, 5’-GCAGGTACCCCTTGGGCCCCACCCCTGGGCCT-3’
ID:231 错误
Report for CCDS5831.1 (current version) CCDS Status Species Chrom. Gene CCDS Release 17 NCBI Ensembl Annotation Annotation Links Release Release 106 76 5831.1 Public Homo sapiens 7 AKR1B1 CCDS Sequence Data Blue highlighting indicates alternating exons. Red highlighting indicates amino acids encoded across a splice junction. Nucleotide Sequence (951 nt):
ATGGCAAGCCGTCTCCTGCTCAACAACGGCGCCAAGATGCCCATCCTGGGGTTGGGTACCTGGAAGTCCCCTCCAGGGCAGGTGACTGAGGCCGTGAAGGTGGCCATTGACGTCGGGTACCGCCACATCGACTGTGCCCATGTGTACCAGAATGAGAATGAGGTGGGGGTGGCCATTCAGGAGAAGCTCAGGGAGCAGGTGGTGAAGCGTGAGGAGCTCTTCATCGTCAGCAAGCTGTGGTGCACGTACCATGAGAAGGGCCTGGTGAAAGGAGCCTGCCAGAAGACACTCAGCGACCTGAAGCTGGACTACCTGGACCTCTACCTTATTCACTGGCCGACTGGCTTTAAGCCTGGGAAGGAATTTTTCCCATTGGATGAGTCGGGCAATGTGGTTCCCAGTGACACCAACATTCTGGACACGTGGGCGGCCATGGAAGAGCTGGTGGATGAAGGGCTGGTGAAAGCTATTGGCATCTCCAACTTCAACCATCTCCAGGTGGAGATGATCTTAAACAAACCTGGCTTGAAGTATAAGCCTGCAGTTAACCAGATTGAGTGCCACCCATATCTCACTCAGGAGAAGTTAATCCAGTACTGCCAGTCCAAAGGCATCGTGGTGACCGCCTACAGCCCCCTCGGCTCTCCTGACAGGCCCTGGGCCAAGCCCGAGGACCCTTCTCTCCTGGAGGATCCCAGGATCAAGGCGATCGCAGCCAAGCACAATAAAACTACAGCCCAGGTCCTGATCCGGTTCCCCATGCAGAGGAACTTGGTGGTGATCCCCAAGTCTGTGACACCAGAACGCATTGCTGAGAACTTTAAGGTCTTTGACTTTGAACTGAGCAGCCAGGATATGACCACCTTACTCAGCTACAACAGGAACTGGAGGGTCTGTGCCTTGTTGAGCTGTACCTCCCACAAGGATTACCCCTTCCATGAAGAGTTTTGA
Translation (316 aa):
MASRLLLNNGAKMPILGLGTWKSPPGQVTEAVKVAIDVGYRHIDCAHVYQNENEVGVAIQEKLREQVVKREELFIVSKLWCTYHEKGLVKGACQKTLSDLKLDYLDLYLIHWPTGFKPGKEFFPLDESGNVVPSDTNILDTWAAMEELVDEGLVKAIGISNFNHLQVEMILNKPGLKYKPAVNQIECHPYLTQEKLIQYCQSKGIVVTAYSPLGSPDRPWAKPEDPSLLEDPRIKAIAAKHNKTTAQVLIRFPMQRNLVVIPKSVTPERIAENFKVFDFELSSQDMTTLLSYNRNWRVCALLSCTSHKDYPFHEEF
SCC+GFP
CGGGATCCGCCACCATGATCTCCACCCGCAGTCCTCGCCCCTTCAATGAGATCCCCTCTCCTGGTGACAATGGCTGGCTAAACCTGTACCATTTCTGGAGGGAGACGGGCACACACAAAGTCCACCTTCACCATGTCCAGAATTTCCAGAAGTATGGCCCGATTTACAGGGAGAAGCTCGGCAACGTGGAGTCGGTTTATGTCATCGACCCTGAAGATGTGGCCCTTCTCTTTAAGTCCGAGGGCCCCAACCCAGAACGATTCCTCATCCCGCCCTGGGTCGCCTATCACCAGTATTACCAGAGACCCATAGGAGTCCTGTTGAAGAAGTCGGCAGCCTGGAAGAAAGACCGGGTGGCCCTGAACCAGGAGGTGATGGCTCCAGAGGCCACCAAGAACTTTTTGCCCCTGTTGGATGCAGTGTCTCGGGACTTCGTCAGTGTCCTGCACAGGCGCATCAAGAAGGCGGGCTCCGGAAATTACTCGGGGGACATCAGTGATGACCTGTTCCGCTTTGCCTTTGAGTCCATCACTAACGTCATTTTTGGGGAGCGCCAGGGGATGCTGGAGGAAGTAGTGAACCCCGAGGCCCAGCGATTCATTGATGCCATCTACCAGATGTTCCACACCAGCGTCCCCATGCTCAACCTTCCCCCAGACCTGTTCCGTCTGTTCAGGACCAAGACCTGG