br Materials and methods br Results and discussion Iron
Materials and methods
Results and discussion Iron (II)/2-oxoglutarate (2-OG)-dependent oxygenases ,  catalyze oxidative reactions of a range of metabolic processes including the hydroxylation of l-proline and l-lysine residues during the post-translational modification of collagen. Mori et al.  purified proline 3-hydroxylase from Streptomyces sp. strain TH1, and its structural gene was cloned. The detected DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158 . The enzyme hydroxylated free l-proline to cis-3-hydroxy-l-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase. Proline trans-4-hydroxylase (P-4-H) from the etamycin producer Streptomyces griseoviridus was identified and shown that the purified enzyme to be a 2-OG-dependent oxygenase . Proline trans-4-hydroxylase and proline cis-3-hydroxylase (P-3-H) have been isolated from Streptomyces and Bacillus sp. , , respectively. The former is used in a recombinant process for the commercial preparation of trans-4-hydroxyproline, which is a starting material for the synthesis of pharmaceuticals . Proline 3-hydroxylase has been purified from Streptomyces sp. strain TH1. Its structural gene has been cloned. Using the amino rosmarinic acid sequence encoded by the TH1 proline-3-hydroxylase gene, a series of synthetic oligonucleotides were designed and synthesized by us. These oligonucleotides were then assembled and PCR amplified to form a single double-stranded DNA fragment that contained the coding sequence for the TH1 proline-3-hydroxylase. We have constructed the 873bp proline-3-hydroxylase (P3H) gene as described in Section 2 and expressed the corresponding protein in E. coli. The nucleotide sequence of the synthetic P3H gene differed from that of the TH1 P3H gene because it was optimized for the expression in recombinant E. coli. The optimized nucleotide sequence (sense strand) for the synthetic P3H gene sequence encodes the exact same amino acid sequence as the native TH1 P3H gene with a calculated molecular mass of 33,154Da (Fig. 1). The DNA sequence revealed that a single nucleotide had been altered during construction of the synthetic P3H gene causing amino acid #219 to become a cysteine, rather than the original serine. A subsequent DNA sequencing reaction revealed that the same error was present in the cloning construct pZERO2.1+P3H and it had carried over into the expression construct pBMS2000+P3H. Enzyme assays of protein preparations from the synthetic P3H expression strain performed in the same time frame revealed that this change did not affect enzyme activity, so it was not corrected. The designation of this strain was BL21(DE3)/pBMS2000+P3Hcys to reflect this discrepancy. This recombinant strain was assigned the number SC16497. Recombinant E. coli expression strain BL21(DE3)/pBMS2000+P3Hcys (SC 16497) was grown in shake flasks at 30°C in MT5 medium and the cells were collected. The cells were disintegrated by sonication and the soluble protein sample was separated by electrophoresis using a sodium dodecyl sulfate (SDS) polyacrylamide gel. A protein molecular weight standard (SeeBluePlus2, Invitrogen) was loaded on the same gel to estimate the molecular weight of the E. coli proteins. Cell samples prepared following IPTG induction of BL21(DE3)/pBMS2000+P3Hcys, displayed a strongly overexpressed protein with an apparent molecular weight of 33kDa, consistent with the expected molecular weight of the desired P3H protein. This overexpressed protein was not present in the expression strain prior to IPTG induction and was also not present in the negative control culture. A comparison of the various time points revealed that the overexpressed protein increased over time with maximal expression noted after an overnight (∼16h) expression period. A comparison of the total protein versus soluble protein samples showed that approximately 50% of the overexpressed protein was found in the soluble fraction.