Jack E. Dixon

Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.

A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells

Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay. Heating the cell extract to 60 degrees C for 10 min completely prevented the interference and slowed the hydrolysis of acetyl-CoA. Substantially higher CAT activities were observed when the extract was heat treated in the presence of EDTA prior to enzyme assay for most cell lines tested. This simple reliable method makes possible the accurate assessment of CAT activities in different cell lines. These observations are particularly pertinent to investigators studying tissue-specific gene expression.

A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis

A procedure is described which provides high yields of pyridylethylated cysteine during gas-phase sequencing of peptides. The method decreases transfer losses by reducing the number of transfer steps required for reduction, alkylation, prior to sequencing a peptide. Proteins bound to polybrene-coated glass-fiber filters used in a gas-phase sequenator may be reduced, pyridylethylated, and sequenced directly on the filter. Reduction of cystine-containing peptides is performed using the nonnucleophilic reductant, tributylphosphine [U. T. Rüegg and J. Rudinger (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 47, pp. 111-116, Academic Press, New York] with concomitant alkylation by 4-vinylpyridine in a buffer soluble in the organic phase. Excess reagents and the buffer are removed by drying the filter and briefly washing with chlorobutane. No N-alkylation is apparent under the conditions described, nor are any amino acid side chains modified. The procedure affords high yields and is particularly useful when subnanomole levels of material must be reduced and alkylated.

Catabolism of neuropeptides by a brain proline endopeptidase.

Summary The substrate specificity of a rat brain proline endopeptidase has been examined with a variety of peptides having as few as 3 and as many as 30 amino acids. Each peptide is selectively cleaved on the carboxyl side of proline. The products of the reaction were separated by thin layer electrophoresis and identified by their amino acid compositions. Proteins such as reduced and alkylated serum albumin and denatured myoglobin were not substrates for the enzyme. The time course for hydrolysis of luteinizing hormone-releasing hormone and human angiotensin II demonstrated that both peptides were selectively and quantitatively cleaved at the expected site.

A new approach for detection and assignment of disulfide bonds in peptides

A new procedure is described for identification of disulfide bonds in peptides by fast atom bombardment mass spectrometry (FABMS). Prolonged bombardment of a disulfide-containing peptide in solution by a high-energy xenon beam results in gradual reduction of the disulfide bond. The reduction is the result of reaction intermediates initially produced by the xenon beam. The method for characterization of interchain disulfide bonds is based on the increase in the relative intensity of the pseudomolecular ions of the reduced peptides with a simultaneous decrease in the relative intensity of the protonated molecular ion of the oxidized peptide. This information allows one to identify peptide fragments covalently linked via intermolecular disulfide bonds. The intrachain disulfide bonds are identified by the increase in the relative intensity of the protonated molecular ion of the reduced peptide, relative to the intensity of the protonated molecular ion of the oxidized peptide. These results indicate that this method can be used to detect disulfide bonds of peptides and provides unambiguous information regarding disulfide bond assignment in peptides. Approximately 1 nmol of sample is required.

Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli☆

We have constructed a full-length cDNA that encodes soybean seed lipoxygenase L-1 and have expressed it in Escherichia coli. This gene was inserted into a pT7-7 expression vector, containing the T7 RNA polymerase promoter. E. coli, strain BL21 (DE3), which carries the T7 promoter in its genome, was transfected with the plasmid. Expression of this gene when the cells were cultured at 37 degrees C yielded polypeptide that was recognized by anti-L-1 antibody, but had very little lipoxygenase activity. Yields of active enzyme were markedly increased when cells were cultured at 15-20 degrees C. When ethanol, which has been reported to be an excellent elicitor of heat-shock proteins in E. coli, was also present at a level of 3% the yield was further increased by 40%. Under optimum conditions 22-30 mg of soluble active enzyme was obtained per liter of culture.

Inhibitors of a rat brain enkephalin aminopeptidase.

Abstract: Eight protease inhibitors of microbiological origin were examined as potential inhibitors of a homogeneous rat brain enkephalin aminopeptidase. Bestatin [(2S,3R)‐3‐amino‐2‐hydroxy‐4‐phenylbutanoyl]‐l‐leucine and analogs of bestatin having basic, acidic, and other neutral amino acids substituted for the Leu residue exhibited inhibition constants ranging from 3.3 ± 10−5 to 8.3 ± 10−8m. The best inhibitor had a positively charged amino acid (Lys) substituted for Leu. A series of phenylalanyl dipeptides were examined as substrates with the aminopeptidase. The amino acid residue on the carboxyl side of the peptide bond undergoing cleavage was varied systematically in the dipeptides to include neutral, acidic, and basic residues. Again, a positively charged amino acid (Arg) adjacent to the bond undergoing scission was kinetically preferred. These results may be used to design highly specific inhibitors of the enkephalin aminopeptidase.

De novo purine nucleotide biosynthesis: cloning, sequencing and expression of a chicken PurH cDNA encoding 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase-IMP cyclohydrolase

The purH cDNA, encoding 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) transformylase-inosine monophosphate cyclohydrolase (ATIC), was cloned by functional complementation of an Escherichia coli purH mutant using a chicken liver cDNA expression library. This represents the first report of the cloning of any eukaryotic ATIC-encoding cDNA (PurH). The avian ATIC mRNA is 2.3 kb long and encodes a protein with an Mr of 64,422. The deduced amino acid sequence is 36% identical to the bacterial purH-encoded enzymes from Bacillus subtilis and E. coli. The avian cDNA was expressed as a glutathione S-transferase (GST) fusion protein that was purified in a single step by affinity chromatography. A novel vector was employed which permits rapid and highly efficient cleavage of the GST fusion protein yielding 10 mg of purified PurH product per liter of bacterial culture. Km values were determined with the purified fusion protein utilizing AICAR and (6-R)N10-formyl-tetrahydrofolate as substrates. These values compare favorably with the isolated avian enzyme, supporting the idea that kinetic, as well as other physical properties of the recombinant fusion protein are similar to the native avian enzyme. Large quantities of purified enzyme and the ability to generate site-directed mutations should make mechanistic studies possible. The recombinant enzyme also affords a simple and reliable approach to identifying new antifolates.

Characterization, kinetics and comparative properties of thiol:protein disulfide oxidoreductase

Abstract An enzyme capable of catalyzing thiol:protein disulfide exchange with glutathione and insulin has been purified to apparent homogeneity as judged by disc electrophoresis, electrophoresis with sodium dodecyl sulfate, equilibrium ultracentrifugation and amino acid sequence analysis of the glycoprotein. The isoelectric point of 4.10 and other physicochemical properties are somewhat different from those of similar enzymes described by other investigators, although there are similarities in substrate specificity and kinetic parameters. The solubilized enzyme catalyzes disulfide interchange with glutathione as well as with a variety of other thiol-containing compounds. Although insulin is the best protein substrate tested, vasopressin, oxytocin, and ribonuclease are also utilized. In the presence of glutathione, the enzyme catalyzes the restoration of the active conformation of “scrambled” ribonuclease. Catalysis depends on an active cysteine residue which can be alkylated/inactivated only in the presence of thiol-containing compounds.

Distribution of carboxypeptidase H messenger RNA in rat brain using in situ hybridization histochemistry: implications for neuropeptide biosynthesis

Carboxypeptidase H is an exopeptidase which is highly specific for C-terminal basic amino acids and thought to play a role in neuropeptide biosynthesis. The distribution of carboxypeptidase H mRNA was examined in adult rat brain using in situ hybridization histochemistry. Enzyme transcripts were detected in all major brain areas. Very high levels of carboxypeptidase H mRNA were found in the hippocampus associated with pyramidal cells. Other brain regions showed varied levels of labelling, ranging from high levels in the magnocellular cells of hypothalamic nuclei to very low levels in the striatum. The appearance of enzyme transcripts throughout brain supports a role for this enzyme in the biosynthesis of many neuropeptides. The expression of transcripts in certain ventricular ependymal cells identified them as a new potential peptidergic cell type. The variations in levels of expression of carboxypeptidase H mRNA may reflect differences in peptidergic activity in different neuronal systems.