Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein

Recombinant human parathyroid hormone (hPTH)-(1-84) was obtained from Escherichia coli using a cleavable fusion protein strategy. The fusion protein contains residues 1-138 of human growth hormone as the amino-terminal region and residues 1-84 of hPTH as the carboxyl-terminal region. A 7-residue linker containing the recognition/cleavage sequence of the site-specific blood coagulation protease activated factor X (factor Xa) joins the two regions. Intact hPTH-(1-84) is released from this fusion protein by cleavage in vitro with factor Xa. The fusion protein was produced at a high level and formed inclusion bodies which allowed it to be easily purified by low speed centrifugation, with a yield of approximately 50 mg/liter of culture. After factor Xa cleavage and high performance liquid chromatography purification, highly purified hPTH was obtained, with a final yield of 1.5-3 mg/liter. Physical and biological characterization of the purified hormone demonstrated that it was intact and active hPTH-(1-84).     

Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli.

The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli. Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate. The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations. However, under all conditions tested, only partial (less than 5%) renaturation was achieved. A second attempt was made using a vector with the trp-lac hybrid promoter. In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography. Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture. The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle. At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity.     

Enhanced ELISA: how to measure less than 10 picograms of a specific protein (immunoglobulin) in less than 8 hours

In this paper we outline a flexible and rapid method to measure picogram quantities of isotype-specific immunoglobulin (Ig), including IgE. Only readily or commercially available reagents are required: isotype-specific, anti-human Ig murine monoclonal antibodies (Mab) to coat microtiter plates, polyclonal alkaline phosphatase-coupled isotype-specific F(ab)'2 or Fab' fragments as second antibodies, and an enhanced developing system that amplifies the signal-to-noise ratio of the quantitatively bound second antibody. The procedure is detailed in the appendix to enable easy application, even if one has no previous experience with ELISAs. This system can be used to detect less than 10 picograms of Ig in cultures supernatants of cells that contain mixtures of various Igs and it can be used to detect the product of a single cell producing Ig. This method also will be applicable to measurement of the minute quantities of lymphokines and other biologically active molecules produced in vitro and found in various fluids in vivo.     

The T-T pyrimidine (6-4) pyrimidinone UV photoproduct is much less mutagenic in yeast than in Escherichia coli.

We have examined the mutagenic properties of the T-T pyrimidine (6-4) pyrimidinone UV photoproduct in Saccharomyces cerevisiae, transforming the yeast cells either with single-stranded vectors that carried this adduct at a unique site or with gapped duplex vectors in which the adduct was located within a 28 nt single-stranded region. In an earlier study with SOS-induced Escherichia coli, we found that this photoproduct is highly mutagenic, specifically generating 3' T-->C substitutions in >85% of replicated molecules, and ascribed this specificity to the formation of a stable guanine-pyrimidinone mispair via hydrogen bonds at N-3 and O-2. In contrast, this adduct is very much less mutagenic in yeast, with 60-70% of molecules being replicated accurately and only 12-20% of them exhibiting 3' T-->C substitutions. The enhanced accuracy may reflect the ability of a yeast DNA polymerase, but not E.coli DNA polymerase III, to trap the adduct in a configuration favorable for the formation of an adenine-pyrimidinone base pair.     

Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis

Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate.     

重组人甲状旁腺激素肽（1-34）在大肠杆菌中的高效融合表达及纯化

人工合成人甲状旁腺激素1-34肽段 (PTH1-34)的cDNA序列,克隆到大肠杆菌蛋白表达载体pThioHis中,获得了高表达菌株。经发酵、破菌、金属鳌合层析、反相层析和凝胶层析后获得了纯度大于95%的hPTH1-34。hPTH1-34肽N端测序和质谱分子量测定结果与天然PTH1-34一致。生物学活性研究表明,hPTH1-34在体外具有刺激腺苷酸环化酶的作用。     

Expression of rat protein phosphatase 2C (IA) in Escherichia coli

A cDNA containing the entire coding sequence of rat type 2C (IA) protein phosphatase was expressed in Escherichia coli. An extract of bacterial cells harboring the recombinant plasmid contained a major (Mr = 41,000 - 43,000) and a minor (Mr = 30,000) protein band; both of these reacted with an anti-type 2C protein phosphatase serum. The size of the major protein band agrees well with that of the 2C phosphatase conceptualized from the cognate cDNA. A Mg2+-dependent protein phosphatase activity was detected in extracts containing the recombinant protein, but not in host cell extracts. Based on these results, it is concluded that the isolated cDNA clone encodes a functional type 2C protein phosphatase.     

Expression of growth hormone-releasing factor analog Leu27GRF(1-44)OH in Escherichia coli: purification and characterization of the expressed protein

A recombinant plasmid has been constructed to direct the synthesis of Leu27GRF(1-44)OH in Escherichia coli as a fusion protein containing a hexa-His tail followed by amino acids 1-99 of interferon-gamma and a methionine residue at the N-terminal. The expression of the 18-kDa fusion protein (H6GAMGRF) was induced by isopropylthiogalactoside treatment and the protein accumulated as insoluble aggregates in inclusion bodies. The protein aggregates were solubilized in 6 M guanidine-HCl and purified directly by affinity chromatography on a Nichelate column. The growth hormone-releasing factor (GRF) moiety was released from the fusion protein by cyanogen bromide cleavage and purified to homogeneity by anion-exchange chromatography followed by reverse-phase chromatography. The identity of the GRF peak was determined by comparing its retention time with that of synthetic Leu27GRF(1-44)OH. The purified material was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and amino-acid analysis. The recombinant-derived product and the synthetic compound showed identical reactivities toward anti-GRF polyclonal antibodies and were essentially equipotent as determined by an in vitro biological assay for growth hormone-releasing activity.     

Expression of enterovirus 70 capsid protein VP1 in Escherichia coli

The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS). High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli.     

Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli.

The covalent intermediate formed during catalysis by the lac Z beta-galactosidase from Escherichia coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apo-enzymes have been determined. The intermediate so formed turns over only very slowly (t1/2 = 11.5 h) resulting in reactivation of the enzyme. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous beta-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M., and Legler, G. (1984) Eur. J. Biochem. 138, 527-531) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.     

Expression of synthetic human angiogenin gene in E. coli in cleaved beta-galactosidase-angiogenin protein

A synthetic gene coding for human angiogenin was cloned in pUR290 plasmid in frame with beta-galactosidase, both parts of the resultant fused protein being joined through an Asp-Pro sequence. The fused protein, synthesised in E. coli cells upon IPTG induction and isolated as inclusion bodies, possessed angiogenic activity on the chick chorioallantois membrane and was cleaved upon acid treatment to yield free angiogenin.     

Expression and purification of uniformly (15)N-labeled amyloid beta peptide 1-40 in Escherichia coli

For biophysical studies using heteronuclear NMR analysis of amyloid beta peptide, construction of an efficient and high yield expression system of uniformly isotopic labeled amyloid beta peptide is desirable. Here we succeeded in obtaining (15)N-labeled amyloid beta 1-40 expressed by attachment to hen egg white lysozyme as a fusion protein.     

Beta-galactosidases (Escherichia coli) with double substitutions show that Tyr-503 acts independently of Glu-461 but cooperatively with Glu-537

Beta-galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed. Control experiments showed that the very low kcat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins. Circular dichroism studies showed that the structures of the enzymes were intact. E461Q/Y503F-beta-galactosidase was inactivated in an "additive" manner. This indicated that Glu-461 and Tyr-503 act independently in catalysis. Because these residues are at opposite sides of the active site and act in different steps, this is expected. E537D/Y503F-beta-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent beta-galactosidases. This showed that Glu-537 and Tyr-503 interact cooperatively on the same step. This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose.     

Expression of gonococcal transferrin-binding protein 1 causes Escherichia coli to bind human transferrin.

The gene for gonococcal transferrin-binding protein 1 (TBP1) was cloned behind an inducible promoter in Escherichia coli. The resultant strain was capable of binding human transferrin with the same specificity as that of the gonococcus. E. coli expressing TBP1 did not internalize transferrin-bound iron or grow on transferrin as a sole iron source.     

β-Galactosidases (Escherichia coli) with Double Substitutions Show That Tyr-503 Acts Independently of Glu-461 but Cooperatively with Glu-537

β-Galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed. Control experiments showed that the very low k _cat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins. Circular dichroism studies showed that the structures of the enzymes were intact. E461Q/Y503F-β-galactosidase was inactivated in an “additive” manner. This indicated that Glu-461 and Tyr-503 act independently in catalysis. Because these residues are at opposite sides of the active site and act in different steps, this is expected. E537D/Y503F-β-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent β-galactosidases. This showed that Glu-537 and Tyr-503 interact cooperatively on the same step. This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose.     

Mutation spectra of N-ethyl-N''-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli

Summary DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GCAT transitions, 4/30 were ATGC transitions, 3/30 were ATTA transversions, and 1/30 was an ATCG transversion. We observed that 37/40 HENU-induced mutations were GCAT transitions and that the remaining 3/40 were ATGC transitions. A majority of the GCAT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5-GG(A or T)-3; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the GA changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The ATGC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5-NTC-3. A strand preference was not apparent for these mutations.     

Valency conversion in the type 1 fimbrial adhesin of Escherichia coli

FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells -- functions readily accomplished by the E. coli-expressing mannotriose-specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH-FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose-only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.     

Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K-12

Using a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli K1 strain to an E coli K-12 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to "authentic" protein K when chymotryptic peptides of 125I-labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ-473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.     

Expression of a proline-enriched protein in Escherichia coli.

The feasibility of expressing repeated synthetic codons in bacterial cells was demonstrated by showing that repeated codons for proline were expressed in Escherichia coli. Recombinant DNA technology was used to clone synthetic polydeoxyguanylate:polydeoxycytidylate into the PstI site of plasmid pBR322. Recombinant plasmid pGC139 was shown by means of HaeIII restriction digestion to contain approximately 41 cloned base pairs; the cloned sequence was expressed as a fusion to an ampicillinase protein. The resulting protein, enriched in proline, was expressed from plasmid pGC139 in E. coli maxicells. Extension of this technology could lead to improvement in the production of amino acids and to nutritional enrichment of single-cell protein.