Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter.

Green fluorescent protein (GFP) was used to study the regulation of the galactose-inducible GAL1 promoter in yeast Saccharomyces cerevisiae strains. GFP was cloned into the pGAL110 vector and transformed into the yeast strains. Time course studies comparing culture fluorescence intensity and GFP concentration were conducted along with on-line monitoring of GFP expression. Our results demonstrated that GFP fluorescence could be used as a quantifiable on-line reporter gene in yeast strains. The effect of an integrated GAL10p-GAL4 transcription cassette was investigated. Induction time studies showed that there was no significant difference in GFP expression level by adding galactose at different culture times. A wide range of galactose concentrations was used to study the initial galactose concentration effect on GFP expression kinetics. A minimum of 0.05 g/L galactose doubled the GFP fluorescence signal as compared to the control, whereas 0.1 g/L gave the highest specific GFP yield. A simple analytical model was proposed to describe GFP expression kinetics based on the experimental results. In addition, this GFP-based approach was shown to have potential use for high-throughput studies. The use of GFP as a generic tool provided important insights to the GAL expression system and has great potential for further process optimization applications.

The self-association and fibronectin-binding sites of fibulin-1 map to calcium-binding epidermal growth factor-like domains.

Fibulin-1 is a modular glycoprotein with amino-terminal anaphylatoxin-like modules followed by nine epidermal growth factor (EGF)-like modules and, depending on alternative splicing, four possible carboxyl termini. Fibulin-1 has been shown to self-associate as well as to bind calcium, fibronectin (FN), laminin, nidogen, and fibrinogen. To map ligand-binding sites within fibulin-1, polypeptides corresponding to various regions of fibulin-1 were expressed recombinantly and evaluated for their capacity to bind calcium, FN, or fibulin-1. A calcium-binding site(s) was mapped to EGF-like modules 5-9. A fibulin-1 self-association site was localized to EGF-like modules 5 and 6 (amino acid residues 356-440), as was a binding site for FN. The self-association interaction mediated by this pair of modules involved calcium since divalent cation chelators reduced the binding affinity of the interaction. By contrast, FN binding to EGF-like modules 5 and 6 was unaffected by the presence of divalent cation chelators. It can be concluded that EGF-like modules 5 and 6 bind calcium and mediate homotypic interaction between EGF-like modules 5 and 6 present in different fibulin-1 molecules and heterotypic interaction between EGF-like modules 5 and 6 and type III repeats 13 and 14 in FN. While additional binding sites for calcium or FN were not detected, another fibulin-1 self-association site was found within amino acid residues 30-173. However, unlike the self-association site in EGF-like modules 5 and 6, which was functional in the native protein, the amino-terminal site was cryptic and revealed only after the protein was denatured.

Adenine quantitation in yeast extracts and fermentation media and its relationship to protein expression and cell growth in adenine auxotrophs of Saccharomyces cerevisiae.

We have described a method to reliably measure the free adenine content of yeast extract powders or the adenine concentrations found in chemically-defined and complex fermentation samples. This method relies on the selective precolumn derivatization of adenine with chloroacetaldehyde to form the fluorescent adenine adduct 1,N6-ethenoadenine. The derivatized adenine can then be resolved from other components found in samples with reverse phase HPLC and selectively monitored with fluorescence. This method was then used to study the adenine nutritional requirements of adenine auxotrophs of recombinant Saccharomyces cerevisiae. The adenine content of individual yeast extract powders was examined in relation to the cell mass (dry cell weight, DCW) achieved in culture media formulated with these powders. A general increase in DCW was observed with increasing adenine concentration in the yeast extract. Conversely, we observed that as adenine concentration increased in complex media the expression levels of a heterologous protein decreased. This method also allowed us to examine the adenine/DCW ratio in both steady-state continuous culture and batch culture. In both cases, the total in vivo adenine content as measured by the amount of adenine utilized from the culture media was estimated to be ca. 25-40 mg/g DCW. However, data suggest that this value is in excess of what is strictly required for cell growth and represents the quantity of adenine required to saturate intracellular pools of adenine or adenine metabolites. A minimum requirement for cell growth is at least as low as 12.5 mg of adenine/g of cells.

Site-directed mutagenesis of residues in a conserved region of bovine aspartyl (asparaginyl) beta-hydroxylase: evidence that histidine 675 has a role in binding Fe2+.

The roles in catalysis of several residues in bovine aspartyl (asparaginyl) beta-hydroxylase that are located in a region of homology among alpha-ketoglutarate-dependent dioxygenases were investigated using site-directed mutagenesis. Previous studies have shown that when histidine 675, an invariant residue located in this highly conserved region, was mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has been undertaken to define the catalytic role of this essential residue. The partial hydroxylase activity observed with some amino acid replacements for histidine 675 correlates with the potential to coordinate metals and not with size, charge, or hydrophobic character. Furthermore, the increase in Km for Fe2+ observed with the H675D and H675E mutant enzymes can account for their partial activities relative to wild type. No significant changes in the Km for alpha-ketoglutarate (at saturating Fe2+) or Vmax were observed for these mutants. These results support the conclusion that histidine 675 is specifically involved in Fe2+ coordination. Further site-directed mutagenesis of other highly conserved residues in the vicinity of position 675 demonstrates the importance of this region of homology in catalysis for Asp (Asn) beta-hydroxylase and, by analogy, other alpha-ketoglutarate-dependent dioxygenases.

Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae.

We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-microm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-microm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1).

Structural integrity of the gamma-carboxyglutamic acid domain of human blood coagulation factor IXa Is required for its binding to cofactor VIIIa.

This report describes the analysis of a novel mutant human factor IX protein from a patient with hemophilia B (factor IX activity <1%; factor IX antigen 45%). Enzymatic amplification of all eight exons of the factor IX gene followed by direct sequence analysis reveals a single nucleotide change (a guanine --> adenine transition) in exon 2 at nucleotide 6409 which results in a glycine --> arginine substitution at amino acid 12 in the gamma-carboxyglutamic acid rich (Gla) domain of the mature protein. Factor IX was isolated by immunoaffinity chromatography from plasma obtained from the proband. The purified protein is indistinguishable from normal factor IX by polyacrylamide gel electrophoresis. Characterization of the variant in purified component assays reveals that it is activated normally by its physiologic activator factor XIa, but its phospholipid-dependent activation by the factor VIIa-tissue factor complex is diminished. In the presence of phospholipid and 5 mM Ca2+, the activities of variant and normal plasma-derived factor IX are similar; however, in the presence of activated factor VIIIa (intrinsic tenase complex), the normal augmentation of the cleavage of the specific substrate of factor IX, factor X, is not observed. The determination of the association constants for normal and variant factor IXa with factor VIIIa shows that the affinity of the activated variant factor IX for the cofactor factor VIIIa is 172-fold lower than normal. Competition studies using active site-inactivated factor IXas in the intrinsic tenase complex confirm that the defect in the variant protein is in its binding to factor VIIIa. We conclude that the structural integrity of the Gla domain of human factor IX is critical for the normal binding of factor IXa to factor VIIIa in the intrinsic tenase complex. In addition, a glycine at amino acid 12 is necessary for normal activation of factor IX by the factor VIIa-tissue factor complex.

A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases.

The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC 1.14.11.16) specifically hydroxylates one aspartic or asparagine residue in certain epidermal growth factor-like domains of a number of proteins. The expression in Escherichia coli, purification, characterization of a fully active catalytic domain, and evidence for the identification of an active-site region of this enzyme are described. Sequence alignment analyses among the vertebrate alpha-ketoglutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant aspartyl (asparaginyl) beta-hydroxylase involved in substrate binding and/or catalysis. Based upon these studies, an alignment of the C-terminal regions of prolyl and lysyl hydroxylase and of aspartyl (asparaginyl) beta-hydroxylase is proposed. When histidine-675, an invariant residue located in a region of homology within this alignment, was mutated to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H675A), no enzymatic activity was detected. Chemical modification studies show that the wild-type protein is protected from iodo[14C]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.

Invertebrate aspartyl/asparaginyl beta-hydroxylase: potential modification of endogenous epidermal growth factor-like modules.

An invertebrate alpha-ketoglutarate-dependent aspartyl/asparaginyl beta-hydroxylase, which posttranslationally hydroxylates specific aspartyl or asparaginyl residues within epidermal growth factor-like modules, was identified, partially purified and characterized. Preparations derived from two insect cell lines catalyzed the hydroxylation of the expected asparaginyl residue within a synthetic epidermal growth factor-like module. This activity was found to be similar to that of the purified mammalian aspartyl/asparaginyl beta-hydroxylase with respect to cofactor requirements, stereochemistry and substrate sequence specificity. Furthermore, recombinant human C1r, expressed in an insect cell-derived baculovirus expression system, was also found to be hydroxylated at the expected asparaginyl residue. Thus, these results establish the potential for invertebrate aspartyl/asparaginyl hydroxylation. Since several invertebrate proteins known to be required for proper embryonic development contain a putative consensus sequence that may be required for hydroxylation, the studies presented here provide the basis for further investigations concerned with identifying hydroxylated invertebrate proteins and determining their physiologic function.

cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.

Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily.

The majority of human urinary stones are primarily composed of calcium salts. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Inhibitors are among the multiple factors that may influence the complex process of urinary stone formation. We have isolated an inhibitor of calcium oxalate crystal growth from human urine by monoclonal antibody immunoaffinity chromatography. The N-terminal amino acid sequence and acidic amino acid content of this aspartic acid-rich protein, uropontin, are similar to those of other pontin proteins from bone, plasma, breast milk, and cells. The inhibitory effect of uropontin on calcium oxalate crystal growth in vitro supports the concept that pontins may have a regulatory role. This function would be analogous to that of other members of the aspartic acid-rich protein superfamily, which stereospecifically regulate the mineralization fronts of calcium-containing crystals.

beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein.

Among the vitamin K-dependent plasma proteins, only protein S contains the post-translationally modified amino acid erythro-beta-hydroxyasparagine (Hyn). Protein S also contains erythro-beta-hydroxyaspartic acid (Hya). The function of these unusual amino acids, located in the epidermal growth factor-like domains, is unknown. To determine if these post-translational modifications contribute to the functional integrity of human protein S (HPS), recombinant human protein S lacking Hya and Hyn (rHPSdesHya/Hyn) was purified from the medium of human kidney 293 cells that were transfected with HPS cDNA and grown in the presence of the hydroxylase inhibitor 2,2'-dipyridyl. Solution-phase equilibrium binding studies revealed that rHPSdesHya/Hyn binds C4b-binding protein (C4BP) in a manner indistinguishable from recombinant HPS and plasma-derived HPS, exhibiting a Kd in the presence of 2 mM CaCl2 of approximately 0.7 nM and a Kd in the presence of 4 mM EDTA approximately 10-fold higher. In a purified component system, rHPSdesHya/Hyn displayed normal anticoagulant cofactor activity in the activated protein C-catalyzed inactivation of coagulation factor Va bound in the prothrombinase complex. In addition, digestion of rHPSdesHya/Hyn with thrombin in the presence of EDTA appeared normal, and 2 mM CaCl2 prevented the cleavage. Together these results suggest that the post-translational modifications of Asn and Asp residues are not necessary for the macromolecular or Ca2+ interactions associated with the anticoagulant and C4BP binding characteristics of HPS.

Bovine liver aspartyl beta-hydroxylase. Purification and characterization.

The alpha-ketoglutarate-dependent dioxygenase, L-asp(L-Asn)-beta-hydroxylase which posttranslationally hydroxylates specific aspartic acid (asparagine) residues within epidermal growth factor-like domains was purified from bovine liver and characterized. A 52-kDa and a 56-kDa species of this enzyme, which accounted for 60 and 30% of the total enzymatic activity, respectively, were purified to apparent homogeneity. Amino-terminal sequence analyses and immunoblots utilizing antisera raised to the intact 52-kDa species as well as to two complementary fragments of this species demonstrated that the 52- and 56-kDa species differ by a 22-amino acid amino-terminal extension. The remaining 10% of the purified enzymatic activity could be accounted for by the presence of immunologically related higher molecular mass forms (56-90 kDa) of L-Asp(L-Asn)-beta-hydroxylase. Strong evidence was obtained from the results of immunoextraction studies that L-Asp(L-Asn)-beta-hydroxylase can be identified with the purified proteins. Kinetic and physical studies suggest that L-Asp(L-Asn)-beta-hydroxylase exists as a monomer with a compact catalytic domain and an extended protease-sensitive amino terminus whose function remains to be determined. Since the purified L-Asp(L-Asn)-beta-hydroxylase hydroxylated both L-Asp- and L-Asn-containing substrates, it is possible that a single enzyme is responsible for the hydroxylation of Asp and Asn residues in vivo.

Partial purification and characterization of bovine liver aspartyl beta-hydroxylase.

In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide based on the structure of the first epidermal growth factor domain in human factor IX (Gronke, R. S., VanDusen, W. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman, P. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3609-3613). The putative enzyme responsible for the posttranslational modification, aspartyl beta-hydroxylase, has been shown to be a member of a class of 2-ketoglutarate-dependent dioxygenases, which include prolyl-4- and lysyl-hydroxylases. In the present study, we describe the solubilization with nonionic detergent of the enzyme from bovine liver microsomes and its purification using DEAE-cellulose followed by heparin-Sepharose. No additional detergent was required during purification. The partially purified enzyme preparation was found to contain no prolyl-4- or lysyl-hydroxylase activity. Using a synthetic peptide based on the structure of the epidermal growth factor-like region in human factor X as substrate, the apparent Km values for iron and alpha-ketoglutarate were 3 and 5 microM, respectively. The enzyme hydroxylated the factor X peptide with the same stereospecificity (erythro beta-hydroxyaspartic acid) and occurred only at the aspartate corresponding to the position seen in vivo. Furthermore, the extent to which either peptide (factor IX or X) was hydroxylated reflected the extent of hydroxylation observed for both human plasma factors IX and X.

Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure of the first growth factor-like domain of human factor IX.

beta-Hydroxylation of aspartic acid is a post-translational modification that occurs in several vitamin K-dependent coagulation proteins. By use of a synthetic substrate comprised of the first epidermal growth factor-like domain in human factor IX and either mouse L-cell extracts or rat liver microsomes as the source of enzyme, in vitro aspartyl beta-hydroxylation was accomplished. Aspartyl beta-hydroxylase appears to require the same cofactors as known alpha-ketoglutarate-dependent dioxygenases. The hydroxylation reaction proceeds with the same stereospecificity and occurs only at the aspartate corresponding to the position seen in vivo. Further purification and characterization of this enzymatic activity should now be possible.