Expression, purification, and characterization of the human squalene synthase: use of yeast and baculoviral systems.

We have cloned and utilized a cDNA corresponding to the human squalene synthase gene to generate active enzyme from yeast and baculoviral expression systems. Expression of human squalene synthase in yeast resulted in production of active enzyme in cellular lysates. The presence of the active human enzyme, however, was insufficient to rescue growth of spores defective in yeast squalene synthase function, suggesting that structural differences in the yeast and human enzymes may affect localization or folding of the protein. Expression of the human enzyme in Sf-9 insect cells after infection with recombinant baculovirus encoding the human squalene synthase gene resulted in detection of substantial enzymatic activity in cell lysate preparations. Following extraction from the Sf-9 cells, the human enzyme was purified to near homogeneity utilizing a series of ion-exchange chromatography steps with an overall yield of purified protein of approximately 5 mg per liter of Sf-9 cell culture. The purified enzyme was characterized through steady-state kinetic and physical measurements and the kinetic constants are consistent with values observed for other squalene synthases. Zaragozic acid C was found to be a competitive inhibitor with respect to farnesyl pyrophosphate and has a Kis value of 250 pM (@ [NADPH] = 5 mM). Inhibition experiments with zaragozic acid C at low (approximately 0.5 x Km) and high (approximately 10 x Km) concentrations of NADPH indicated that the inhibitor does not bind in the enzyme's NADPH binding domain. These studies demonstrate that the human enzyme can be prepared from baculovirus-infected Sf-9 cells in a catalytically active configuration and in sufficient quantities to allow for further biochemical, kinetic, and structural characterization.

Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase.

Yeast squalene synthetase which has been truncated by 24 amino acids at the C-terminus has been overexpressed in Escherichia coli and constitutes approximately 20% of the total soluble cell protein. For the first time, milligram quantities of this essential enzyme in the cholesterol biosynthetic pathway have been purified to near homogeneity by ammonium sulfate precipitation and Mono Q anion-exchange chromatography so that the steady-state rate constants could be measured. A combination of 10% methanol, 10% glycerol, 30 mM octyl-beta-D-glucopyranoside, 0.4% Brij-58, and 1 mM dithiothreitol in 25 mM sodium phosphate, pH 7.4, was essential for the stability and maximal enzyme activity of the near homogeneous enzyme. Kinetic analysis indicated a Km for farnesyl pyrophosphate of 2.5 microM, suggesting fairly tight binding of farnesyl pyrophosphate to truncated yeast squalene synthetase. The turnover number, kcat, for the conversion of farnesyl pyrophosphate to squalene was 0.53 s-1, and the apparent second order rate constant, kcat/Km, was 2.1 x 10(5) M-1 s-1, indicating a relatively slow conversion of farnesyl pyrophosphate to squalene and a low specificity constant for this enzyme. In addition, Km for NADPH and NADH was 0.5 and 3.6 mM, respectively. Moreover, truncated yeast squalene synthetase shows a preference for NADPH over NADH as reflected in the sevenfold higher kcat/Km value for NADPH similar to that for the native enzyme.

Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms.

cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5' end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3' to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II.

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli.

The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.

The alpha and beta chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: characterization of enzymatic activities.

Reverse transcriptase of the avian sarcoma and leukosis retroviruses is a heterodimer composed of a 63-kDa alpha and a 95-kDa beta polypeptide chain, both of which are encoded in the pol gene and are produced by proteolytic processing of a larger precursor. We previously constructed a bacterial expression clone of the entire pol coding region that produces a protein 4 kDa larger than the mature viral beta subunit. By use of this clone and synthetic oligonucleotides to introduce stop codons, two derivatives have been constructed: one that directs synthesis of a protein equivalent to the mature beta subunit and the other that directs synthesis of a protein equivalent to alpha subunit. Predicted amino acid sequences of these proteins differ from their viral counterparts only by an initiator methionine that was added to the N termini for expression in Escherichia coli. Both bacterially expressed proteins exhibit reverse transcriptase activity and appear to function as homodimers. The properties of these proteins resemble those of the viral reverse transcriptase heterodimer; however, the bacterially produced alpha dimer protein could be distinguished from the other proteins by its increased sensitivity to heat inactivation, which also has been reported for the corresponding viral product. These results show that correct folding and expression of enzymatic function does not require formation of a precursor. The alpha and beta clones provide a convenient source of individual pol gene products for further evaluation of their roles in the synthesis and integration of retroviral DNA.

Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain.

Three pol gene products have been identified in avian retroviral particles: the full-length 95-kilodalton (kDa) beta chain of reverse transcriptase and two proteolytic cleavage products of beta, a 63-kDa reverse transcriptase alpha chain derived from the amino terminus of beta and a 32-kDa (pp32) endonuclease from its carboxy terminus. By using molecularly cloned retroviral DNA and synthetic oligonucleotides to introduce initiator ATGs and codons corresponding to the authentic N termini, we constructed two bacterial-expression clones; one clone contains the entire pol gene, and the other contains the region encoding the pp32 domain. A 99-kDa protein was synthesized in Escherichia coli by the full-length clone, and a 36-kDa protein was synthesized by the endonuclease domain clone. The recombinant proteins exceeded the size of both the mature viral beta chain and the pp32, respectively, by approximately 4 kDa. These larger sizes, however, are consistent with predictions from the DNA sequence of the pol gene. Processing of the recombinant pol proteins was examined by using p15 protease purified from virus particles and antisera directed against synthetic peptides corresponding to three domains in pol. Proteolytic digestion of the 99-kDa product with p15 produced a 63-kDa protein that comigrated on polyacrylamide gels with the alpha chain of reverse transciptase and a 36-kDa fragment that comigrated with the endonuclease domain product. Further digestion of the 36-kDa protein yielded a 32-kDa protein that comigrated with viral pp32 endonuclease. Thus, we concluded that two p15-sensitive sites exist in pol. Cleavage at the previously identified site produces alpha, and cleavage at the newly discovered site removes approximately 4 kDa from the C terminus of the primary protein product. Since the 36-kDa protein was also detected in protein isolated from virus particles, it seems probable that processing at the C-terminal site is a normal step in the production of mature beta and pp32 endonuclease products.

recA protein-promoted DNA strand exchange. Effect of ionic strength.

The addition of from 80 to 120 mM NaCl to a recA protein-promoted DNA strand exchange results in a significant increase in both the initial rate and extent of reaction. This increase results from the formation of a relatively stable recA protein-single-stranded DNA complex that is similar to the complex formed in the presence of the single-stranded DNA-binding protein. In addition to increasing the rate and extent of strand exchange, the amount of recA protein and ATP hydrolysis needed to promote these reactions efficiently is reduced. However, single-stranded DNA-binding protein is significantly more effective than addition of 100 mM NaCl in promoting stable complex formation and stimulating strand exchange. These findings support the hypothesis that a stable recA protein-single-stranded DNA complex is required for efficient DNA strand exchange.

An unpaired 3' terminus stimulates recA protein-promoted DNA strand exchange.

In the presence of 100 mM NaCl, the efficient exchange of strands between a circular single strand and an homologous DNA duplex promoted by the recA and single-stranded DNA binding proteins of Escherichia coli requires an unpaired 3' terminus. Of the duplex DNAs tested, only those with 4 unpaired bases at the 3' termini are effective. Without added NaCl, strand exchange proceeds efficiently with all duplex DNA termini examined including a nicked circular duplex. Thus, at approximately physiological salt concentrations, factors in addition to the recA and single-stranded DNA binding proteins are needed to promote efficient strand exchange. One such factor may be a DNA helicase(s).

recA protein promoted DNA strand exchange.

recA protein and circular single-stranded DNA form a stable complex in the presence of single-stranded DNA binding protein (SSB), in which one recA protein monomer is bound per two nucleotides of DNA. These complexes are kinetically significant intermediates in the exchange of strands between the single-stranded DNA and an homologous linear duplex. After completion of strand exchange, the recA protein remains tightly associated with the circular duplex product of the reaction and the SSB is bound to the displaced linear single strand. Upon addition of ADP, the recA protein-duplex DNA complex dissociates. RecA protein also interacts with single-stranded DNA in the absence of SSB; however, the amount of recA protein bound is substantially reduced. These findings provide direct physical evidence for the participation of SSB in the formation of the recA protein-single-stranded DNA complexes inferred earlier from kinetic analysis. Moreover, they confirm the ability of recA protein to equilibrate between bound and free forms in the absence of SSB.

On the role of single-stranded DNA binding protein in recA protein-promoted DNA strand exchange.

RecA protein-promoted DNA strand exchange is greatly stimulated by the single-stranded DNA binding protein (SSB) of Escherichia coli. Stimulation is not a consequence of the binding of SSB to excess single-stranded DNA. It results instead from stabilization of recA protein-single-stranded DNA complexes formed in the presence of ATP and SSB. In the presence of SSB, recA protein does not measurably dissociate from these complexes for up to 90 min. However, in its absence, recA protein moves rapidly between two populations of single-stranded DNA, and complete equilibration occurs with t 1/2 of 17 s. Rapid transfer of recA protein to single-stranded DNA occurs during all stages of DNA strand exchange and does not require ATP. The transfer involves an equilibrium between free and bound recA protein rather than a direct redistribution between single-stranded DNA molecules. Thus, SSB prevents dissociation of recA protein from single-stranded DNA, rendering the binding of the recA protein to single-stranded DNA irreversible. Under these conditions, the pairing phase of the strand exchange reaction is accelerated to the point that it is no longer rate-limiting. These results can explain the relative inefficiency of DNA strand exchange in the absence of SSB.

ADP-mediated dissociation of stable complexes of recA protein and single-stranded DNA.

The complete exchange of strands between circular single-stranded and full length linear duplex DNAs promoted by the recA protein of Escherichia coli is dependent upon the hydrolysis of ATP and is strongly stimulated by the single-stranded DNA binding protein (SSB). In the presence of SSB, stable complexes of recA protein and single-stranded DNA are formed as an early step in the reaction. These complexes dissociate when the ADP/ATP ratio approaches a value of 0.6-1.5, depending upon reaction conditions. Thus, ATP hydrolysis never proceeds to completion but stops when 40-60% of the input ATP has undergone hydrolysis. recA protein can participate in a second round of strand exchange upon regeneration of the ATP. While 100-200 mol of ATP are hydrolyzed/mol of heteroduplex base pair formed under standard reaction conditions in the presence of SSB, this value is reduced to 16 at levels of ADP lower than that required to dissociate the complexes. ATP hydrolysis appears to be completely irreversible since efforts to detect exchange reactions using 18O probes have been unsuccessful.

Isolation and characterization of two mutant forms of T4 polynucleotide kinase.

The purification of polynucleotide kinase from Escherichia coli infected by two different mutants in the T4 polynucleotide kinase (pseT) gene is described. The pseT 1 enzyme has virtually no 3' specific phosphatase activity and normal polynucleotide kinase activity. The pseT 47 enzyme has very little phosphatase activity and no kinase activity. However, enzyme isolated from a pseT 1, pseT 47 mixed infection appears to contain heterodimers with considerably more phosphatase activity. Thus, the pseT 47 mutation partially inactivates the phosphatase and totally inactivates the kinase. A study of the action of polynucleotide kinase on plasmid DNAs nicked to give a 3'-phosphate and a 5'-hydroxyl indicates that although the enzyme can catalyze both the removal of the 3'-phosphate and the insertion of a 5'-phosphate, there is no evidence for a concerted reaction involving both activities on the same polypeptide chain.

Independent locations of kinase and 3'-phosphatase activities on T4 polynucleotide kinase.

We have used two chemical modification reagents and three proteases to study the relationship between the two activities of T4 polynucleotide kinase. In each case, conditions were found where one of the two activities of the enzyme could be eliminated without greatly reducing the other. Taken together, these data indicate that the two activities are catalyzed by amino acid residues located in separate active sites on the polypeptide chain. Specific exopeptidase digestion indicates that the kinase activity lies in the NH2-terminal and the phosphatase in the COOH-terminal portion of the polypeptide chain. Partial trypsin digestion produces a 29,000-dalton fragment with no kinase activity and nearly normal 3'-phosphatase activity.