Characterization of C-terminal-truncated urinary metabolites of a recombinant hirudin in rats

Although it has been reported that hirudin was excreted in urine mainly as its nonmetabolized form in humans, dogs, and rabbits, no report has been published about the molecular nature of urinary metabolites in rats. We found that nonmetabolized hirudin could not be detected in rat urine after its i.v. administration and that urinary metabolites of recombinant hirudin CX-397 consisted of at least the following six C-terminal-truncated peptides: CX-397^1–49, CX-397^1–50, CX-397^1–51, CX-397^1–52, CX-397^1–54, and CX-397^1–55, in the ratio of roughly 11, 51, 3, 11, 19, and 5%, respectively. In conclusion, the urinary metabolism of recombinant hirudin in rats is different from that in humans, dogs, and rabbits, suggesting that the handling of hirudin in rat kidney is unique among them.     

Expression of active rat DNA polymerase beta in Escherichia coli

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.     

Co-translational incorporation of trans-4-hydroxyproline into recombinant proteins in bacteria

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (alpha1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins.     

Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2).

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.     

Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli

Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.     

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.     

Identification of the gene encoding transcription factor NusG of Thermus thermophilus.

The nusG gene of Thermus thermophilus HB8 was cloned and sequenced. It is located 388 bp downstream from tufB, which is followed by the genes for ribosomal proteins L11 and L1. No equivalent to secE preceding nusG, as in Escherichia coli, could be detected. The nusG gene product was overproduced in E. coli. A rabbit antiserum raised against the purified recombinant NusG reacted exclusively with one protein band of T. thermophilus crude extracts in Western blot (immunoblot) analyses, and no cross-reaction of the antiserum with E. coli NusG was observed. Recombinant NusG and the reacting T. thermophilus wild-type protein had identical sizes on sodium dodecyl sulfate-polyacrylamide gels. T. thermophilus and E. coli NusG have 45% identical and 22.5% similar amino acids, and similarities between the two proteins are most pronounced in carboxy-terminal regions. The T. thermophilus nusG gene could not rescue a nusG-deficient E. coli mutant strain.     

Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli.

The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.     

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.     

Purification and characterization of the Streptomyces lividans initiator protein DnaA.

The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains. The larger size of the S. lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II. The S. lividans protein was overproduced as a His-tagged fusion protein. The purified protein (isoelectric point, 5.7) has a weak ATPase activity. By DNase I footprinting studies, each of the 17 DnaA boxes (consensus sequence, TTGTCCACA) in the S. lividans oriC region was found to be protected by the DnaA fusion protein. Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif or with amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes. A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S. lividans mutant DnaA proteins with little or no capacity for binding to DnaA boxes. Thus, like in E. coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA. A mutant protein lacking a stretch of acidic amino acids corresponding to domain II is not affected in its DNA binding capacity. Whether the acidic domain II interacts with accessory proteins remains to be elucidated.     

Excretion of human beta-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli

Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage lambda to construct E. coli MBEL-BL101. For the excretion of human beta-endorphin as a model protein, the beta-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-beta-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-beta-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield beta-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of beta-endorphin was obtained.     

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.     

The relA/spoT-Homologous Gene in Streptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.     

Enhanced alpha-amylase production in recombinant Bacillus brevis by fed-batch culture with amino acid control

Fed-batch culture with controlled L-amino acid composition was performed to improve production of a recombinant gene product in Bacillus brevis. The maximum recombinant protein (alpha-amylase) level and specific activity increased from 5.14 kU/mL and 0.77 kU/mg dry cell in conventional fed-batch culture to 12.01 kU/mL and 2.64 kU/mg dry cell, respectively, when L-amino acid concentration was controlled at 5 mM using an asparagine (Asn)- and isoleucine (Ile)-enriched nitrogen source. The L-amino acid concentration in the culture was monitored by an automatic biotech analyzer and controlled at 2-20 mM using a mixture of polypeptone and yeast extract. Although L-amino acid concentrations were controlled at low levels, the alpha-amylase activity increased only 1.3 times compared to an uncontrolled batch culture; accumulation of ammonium ion was not reduced. When L-amino acid was controlled at the high level, more cell mass and less recombinant gene product were produced than in those with low control level. To overcome ammonium ion inhibition, the specific amino acids Asn and Ile were substituted to improve the production of gene product. Addition of these amino acids to a flask culture led to an improvement in the enzyme production level and specific activity to 2.9 and 5.1 times, respectively, as high as that without them. Both the control of amino acids at low concentrations and the enrichment of Asn and Ile were effective for the improvement of recombinant protein production from recombinant B. brevis cells. (c) 1996 John Wiley & Sons, Inc.     

Structure-function relationship of bacterial prolipoprotein diacylglyceryl transferase: functionally significant conserved regions.

The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.     

High level expression and preparation of autonomous Ca2+/calmodulin-dependent protein kinase II in Escherichia coli

The chymotryptic fragment of Ca2+/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active enzyme that phosphorylates a variety of protein substrates in vitro. Although 30K-CaMKII is an often used and powerful tool for protein phosphorylation, the efficient production of catalytically active 30K-CaMKII in Escherichia coli has not yet been successfully realized, probably due to its toxicity in host cells. In this study, we found that a high-level expression of 30K-CaMKII as an insoluble form was attained when the N-terminal 43 amino acid residues of Xenopus CaMKI were fused to the N-terminal end of 30K-CaMKII (CX-30K-CaMKII). The inactive CX-30K-CaMKII thus expressed in E. coli was reactivated by simple denaturation/renaturation processes and purified on a Ni2+-chelating column. The renatured CX-30K-CaMKII exhibited specific activity similar to that of rat brain CaMKII, and phosphorylated various proteins such as histones, myosin light chain, myelin basic protein, and synapsin I, as in case of 30K-CaMKII or purified CaMKII. Thus, CX-30K-CaMKII, an autonomous CaMKII, can be obtained with a simple procedure using E. coli expression system.     

重组水蛭素的纯化和鉴定

本文报导了化学合成的水蛭素基因在酵母细胞中得到表达,井能分泌水蛭素到胞外。将该菌株培养物的上清液经硫酸铵沉淀和Sephadex G-50过滤后,用DEAE-SephadexA-25进行阴离子交换层析,进而用HPLC反相层析,得到表达产物重组水蛭素。经SDS-PAGE,氨基酸序列分析,抗凝血酶活力分析及血浆滴定实验等方法鉴定,证明该基因表达产物与天然水蛭素HV_2相同。     

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.     

Expression of the Saccharomyces cerevisiae PIS gene and synthesis of phosphatidylinositol in Escherichia coli.

Expression of the Saccharomyces cerevisiae PIS gene encoding phosphatidylinositol synthase in Escherichia coli was achieved by inserting its coding sequence into lacZ on pUC8. The fused gene encoded a phosphatidylinositol synthase whose amino-terminal three amino acids had been replaced by the amino-terminal five amino acids of E. coli beta-galactosidase. E. coli cells bearing this recombinant plasmid produced a significant level of phosphatidylinositol synthase in the presence of a lacZ inducer, isopropylthio-beta-D-galactopyranoside. When the culture medium was supplemented with myo-inositol and isopropylthio-beta-D-galactopyranoside, the cells accumulated a substantial amount of phosphatidylinositol in their membranes. When a saturating level of myo-inositol was added, phosphatidylinositol constituted about 4% of the total phospholipids. Phosphatidylinositol accumulation occurred at the expense of phosphatidylglycerol. The ratio of phosphatidylethanolamine to total acidic phospholipids remained constant. The growth rate of phosphatidylinositol-containing E. coli cells did not differ significantly from that of cells with the normal phospholipid composition.