Dihydroorotase from Escherichia coli. Cloning the pyrC gene and production of tryptic peptide maps.

We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.

We have inserted 8 1.7-kilobase pair Escherichia coli DNA fragment containing the l-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking thspyrC plasmid tryptic peptide maps from the two dihydroorotase preparations, preduced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.
Dihydroorotase (~-5,6-dihydroorotate amidohydrolase; EC 3.5.2.3) has been purified previously from a strain of Escherichia coli (ATCC 29477) that contains the chromo6omally derived enzyme as 0.23% of its soluble protein, a 5-8-fold enrichment over wild type (Washabaugh and Collins, 1984). That purification procedure required approximately 1 month to produce 19 mg of enzyme, beginning with frozen E. coli (ATCC 29477) cell paste. We present here procedures for inserting the l-kilobase pair E. colipyrC gene into a high copy number plasmid to produce a strain of E. coli containing elevated levels of dihydroorotase. This strain will allow the convenient preparation of gram quantities of dihydroorotase. We also compare peptide maps from purified pyrC plasmidderived and chromosomally derived enzyme to evaluate the effects of our cloning procedures on the structure of the enzyme.
After our cloning was completed, Jensen et al. (1984) reported the insertion of the E. coli pyrC gene on a 3-kilobase pair DNA fragment from the same Clarke-Carbon collection plasmid (Clarke and Carbon, 1976) into pBR322. This recombinant plasmid, which produced dihydroorotase as approximately 0.5% of the total soluble protein, was used for studies * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Supported by Grant 1T32 ES07141 from the National Institute of Environmental Health Sciences. on the control of gene expression. The work we report here, which uses a smaller E. coli DNA insert, is focused on maximizing the yield of dihydroorotase and establishing that the overproduced enzyme is unaltered.  Table 1, Figs. 1-4, "Discussion," and "Acknowledgments" are presented in miniprint at the end of this paper. The abbreviations used are: TPCK, L-l-tosylamido-2-phenylethyl chloromethyl ketone; HPLC, high performance liquid chromatography; kb, kilobase pair. Miniprint is easily read with the aid of standard magnifiying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-3381, cite the authors, and include a check or money order for $7.60 per set of photocopies. Full size photocopieu are also included in the microfilm edition of the Journal that is available from Waverly Press. ""__"""_""""""""""""""""""""""""""------------  Figure 2 ShoYs t h a t when E. poi?. S t r a i n Xl014a (pyrc-) containing pKC16eyG w a s grown i n modified H56 medium for 4 h a t 3ooc, and then shifted to 42% POP 24 h, dihydroorotase UBB found st high level9 i n the tine inteFYa1 12-24 h.
Our f a i l w e t o produce high levels of dihydmorotase using the PL promoter i n pHE2 lod