Expression of the Methanobacterium thermoautotrophicum hpt gene, encoding hypoxanthine (Guanine) phosphoribosyltransferase, in Escherichia coli

Abstract

The hpt gene from the archaeon Methanobacterium thermoautotrophicum, encoding hypoxanthine (guanine) phosphoribosyltransferase, was cloned by functional complementation into Escherichia coli. The hpt-encoded amino acid sequence is most similar to adenine phosphoribosyltransferases, but the encoded enzyme has activity only with hypoxanthine and guanine. The synthesis of the recombinant enzyme is apparently limited by the presence of the rare arginine codons AGA and AGG and the rare isoleucine AUA codon on the hpt gene. The recombinant enzyme was purified to apparent homogeneity.

FIG. 1

The nucleotide sequence of a 1,119-bp StyI-PstI DNA fragment containing part of orfA and orfB and the complete hpt gene of M. thermoautotrophicum Marburg. The deduced amino acid sequence of the HGPRTase, encoded by hpt, and the two open reading frames are shown above the sequence. The amino acids shown in bold have been verified by N-terminal sequencing of the purified HGPRTase. The potential ribosome binding sites in front of the open reading frames are marked with #. Relevant restriction sites are indicated and double underlined. The transcriptional start sites of the hpt gene, as identified by primer extension (Fig. 2) are marked with arrows, and the putative promoter region is marked with asterisks. Amino acids encoded by infrequently used arginine and isoleucine codons in E. coli are boxed.

FIG. 2

Primer extension analysis of the hpt-specified mRNA. The numbering of the nucleotide sequence is as in Fig. 1, with the coding strand displayed. A 50-μg quantity of RNA prepared from M. thermoautotrophicum was used in each experiment, and results obtained with complete extension mixture (+) and with an extension mixture without reverse transcriptase (as a control) (−) are shown. Lanes G, A, T, and C show the sequences generated using primer 15350 together with plasmid pSAUE 7 as template. The putative transcriptional start sites are marked on the gel with arrows and on the sequence to the left of the gel with asterisks. A putative box A promoter element is boxed on the sequence.

FIG. 3

Sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis of the purification steps of the recombinant HGPRTase from M. thermoautotrophicum. A total of 10 μg of protein was applied at each purification step (Table 1). Lanes: 1, crude extract; 2, streptomycin step; 3, heat denaturation step; 4, DEAE step; 5, GTP-agarose affinity step. The molecular mass markers used were as follows: Phosphorylase b (97.4 kDa); serum albumin (66.2 kDa); ovalbumin (45.0 kDa); carbonic anhydrase (31.0 kDa); soybean trypsin inhibitor (21.5 kDa); and lysozyme (14.5 kDa). The position of the purified HGPRTase is marked with an arrow.

FIG. 4

Growth of E. coli SØ609/pSAUE12 and HGPRTase levels. Effects of IPTG and plasmid pRI952, containing genes that encode tRNAs for the rare codons AGA, AGG, and AUA. Closed symbols indicate growth (optical density at 436 nm [OD₄₃₆]) in control cultures, and open symbols indicate growth after the addition of IPTG. Time of IPTG addition is indicated. Symbols: ○ and ●, SØ609/pSAUE12; ▵ and ▴, SØ609/pSAUE12 + pRI952. Numbers shown above and below the growth curves are HGPRTase levels given as nanomoles per minute per milligram of protein determined in cells isolated at the last point of the growth curve.