Overproduction and purification of McrC protein from Escherichia coli K-12.

The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system. Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al. (J. Bacteriol. 171:1974-1981, 1989).     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.     

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.     

Overproduction of soluble recombinant transglutaminase from <Emphasis Type="Italic">Streptomyces netropsis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Identification of the Soybean Allergenic Protein,Gly m Bd 30K,with the Soybean Seed 34-kDa Oil-body-associated Protein

The soybean allergenic protein, Gly m Bd 30K [Ogawa et al., J. Nutr. Sci. Vitaminol., 37, 555–565 (1991)] which is most strongly and frequently recognized by the IgE antibodies in sera of soybean-sensitive patients with atopic dermatitis, has been characterized. The allergen was isolated from the crude 7S-globulin fraction as an oligomeric form with a molecular weight of more than 3000,000 by gel-filtration chromatography. On two-dimensional gel electrophoresis, the native oligomeric allergen had an isoelectric point of about pH 4.5 and was dissociated into a monomeric form with a molecular weight of about 32,000 by the treatment with sodium dodecyl sulfate and 2-mercaptoethanol. The monomeric allergen had an N-terminal amino acid sequence and amino acid composition identical with those of the soybean seed 34-kDa oil-body-associated protein or the soybean vacuolar protein P34 with close homology to papain-like thiol proteinases [Kalinski et al., J. Biol. Chem., 267, 12068 (1992)]. The identity was further confirmed by the immunological cross-reactivity to the antibodies produced against each of the purified allargen and the 34-kDa oil-body-associated protein. By this observation, Gly m Bd 30K was shown to have about 30% sequence homology with Der pI, a house dust mite allergen that is a thiol proteinase from Dermatophagoides pteronyssius.     

Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens

The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.     

Patatin,a major soluble protein of the potato (Solanum tuberosum L.) tuber is synthesized as a larger precursor

Antibodies were raised against the highly purified glycoprotein patatin. They were used to characterize the product synthesized in a wheatgerm cell-free translation system, programmed with polyadenylated RNA from potato tubers. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that the immunoprecipitated protein had a molecular mass of 43 kDa compared to 40 for the authentic patatin. It is assumed that patatin is synthesized in vivo as a larger precursor which is processed to the mature protein by cleavage of a signal peptide. Our results are in agreement with sequence-analysis data of patatin complementary DNA which indicate a signal peptide of about 23 amino acids (Mignery et al., 1984; Nucleic Acids Res. 12, 7987–8000).Abbreviation Poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Preliminary results were published in Mitteilungsband, Botaniker Tagung in Wien, p. 180, Wien, September 1984     

Isolation and characterisation of porin from the outer membrane of Synechococcus PCC 6301

Pore-forming protein (porin) was isolated from N,N-dimethyl-dodecylaminoxid (LDAO)-extracted outer membranes of Synechococcus PCC 6301 and purified by ion exchange chromatography on DEAE-Sephacel column. The apparent molecular mass on SDS-PAGE was determined to be about 52000. The native porin was reconstituted into black lipid bilayer membranes and showed a single-channel conductance of 5.5 nS in 1 M KCl. The porin was found to be N-terminally blocked. The C-terminal amino acid sequence was identified as Phe-Thr-Phe. Amino acid analysis suggested that the porin protein consists of about 420 amino acid residues, yielding a polarity of 43.6% and a molecular mass of 45000 in contrast to the mobility on SDS-PAGE.Abbreviations DEAE Diethylaminoethyl; M _r, relative molecular mass - LDAO N,N-Dimethyl-dodecylaminoxid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoretogram - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - UTEX Culture Collection of Algae at the University of Texas     

Purification,Characterization, and Gene Analysis of Cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both β-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the β-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to β-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Acidovorax sp. strain SA1 and purification of the enzyme

The gene of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase (i3HBOH) was cloned and sequenced from a poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Acidovorax sp. strain SA1. The i3HBOH gene has 876 nucleotides corresponding to the deduced sequence of 292 amino acids. In this amino acid sequence, the general lipase box sequence (G-X₁-S-X₂-G) was found, whose serine residue was determined to the active sites serine by site-directed mutagenesis. An i3HBOH was purified to electrophoretical homogeneity from SA1. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE. The N-terminal amino acid sequence of the purified enzyme corresponded to the deduced N-terminal amino acid sequence in the cloned i3HBOH gene. This is the first cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase gene to date. Received: 19 October 2001 / Accepted: 7 December 2001     

Purification and biochemical characterization of the complete structure of a proteolytically modified beta-2-microglobulin with biological activity

A modified form of beta-2-microglobulin (beta-2-m) has previously been described to be present in serum from patients suffering from autoimmune diseases, acquired immune deficiency syndrome and small-cell lung cancer [Plesner, T. and Wiik, A. (1979) Scand. J. Immunol. 9, 247-254; Bhalla et al. (1985) Clin. Chem. 31, 1411-1412; Nissen et al. (1984) Clin. Chim. Acta 141, 41-50]. In the present study we describe the purification and characterization of this modified human serum beta-2-m from patients with small-cell lung cancer. Purified urinary beta-2-m was added to the serum samples incubated at 20 degrees C for five days to obtain a higher yield of modified beta-2-m (m-beta-2-m). m-beta-2-m was then purified from serum by gel filtration followed by chromatofocusing of the fractions containing beta-2-m. m-beta-2-m was found to have an apparent molecular mass of 15 kDa and a pI of 5.3 when analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and analytical isoelectric focusing respectively. Amino acid analysis of m-beta-2-m revealed that the protein is missing one lysine residue compared to the composition deduced from the cDNA sequence of beta-2-m. Amino acid sequence analysis showed that m-beta-2-m consists of two polypeptide chains produced by a proteolytic cleavage of beta-2-m in the disulphide loop. After reduction and alkylation of m-beta-2-m the two chains were separated by reverse-phase high-pressure liquid chromatography. By amino acid sequencing, amino acid residues 1-56 and 59-99 were identified in the A and B chains respectively. By comparison of the amino acid composition of m-beta-2-m with the known sequence of beta-2-m it was possible to deduce the existence of a Ser-57 in the A chain. Thus proteolytic cleavage of beta-2-m in the intrachain disulphide loop releases the amino acid Lys-58, which results in a modified form of beta-2-m with a molecular mass of 11,620 Da as determined by amino acid analysis.     

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-D-(1, 4-alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glg B gene

The nucleotide sequence of the glg B gene, coding for branching enzyme (EC 2.4.1.18), was elucidated. It consists of 2181 base pairs specifying a protein of 727 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis that was obtained with the pure protein as well as with the molecular weight determined from sodium dodecyl sulfate-gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal amino acid sequence and the amino acid sequence analysis of various peptides obtained from CNBr degradation of purified branching enzyme.     

E. coli galactose-1-phosphate uridyl transferase: N-terminal and C-terminal sequences

Summary A modified procedure for the purification of E. coli galactose-1-phosphate uridyl transferase (E.C. 2.7.6.12) was developed which reproducibly gives pure enzyme. The purified enzyme was shown to be a dimeric protein with a subunit molecular weight of 41,000 and its amino acid composition and content of free sulfhydryl groups were determined. The N-terminal and C-terminal amino acid sequences were found to be NH₂-thr-gln-phe-asn-pro-val-asp and -ser(val leu)-ala-COOH respectively. This N-terminal sequence allowed the identification of the start of the transferase gene in the DNA sequence determined by GRINDLEY. Furthermore it appears to define a nine base intercistronic region between the epimerase and transferase genes.Abbreviations Cyclic AMP Cyclic adenosine 2¹5¹ monophosphate - DPN Diphosphopyridine nucleotide - UDP Uridine diphosphate - EDTA Ethylene diamine tetra acetic acid - SDS sodium dodecyl sulfate - NEM N-ethylmaleimide     

Isolation and structural studies of the intact scrapie agent protein

Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had greater than 10(11) LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N alpha-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.     

Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.     

Primary structure of the tms and prs genes of Bacillus subtilis

Summary The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis. The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene products remains to be established. The prs gene contains an open reading frame of 317 codons resulting in a subunit M_r of 34828. An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an M_r of 49554. Comparison of the deduced B. subtilis PRPP synthetase amino acid sequence with PRPP synthetases from Escherichia coli and rat liver showed extensive similarity. The deduced Tms amino acid sequence was found to be 43% similar to the deduced amino acid sequence of ecourfl, a gene of E. coli with unknown function.     

Subunit III (Psa-F) of photosystem I reaction center of the C4 dicotyledon Flaveria trinervia

An immunological survey of C3, C4 and C3-C4-intermediate Flaveria species showed that subunit III (PsaF) of the photosystem I reaction center (PSI-RC) is present in all these species. This was confirmed by the isolation of the gene encoding the PSI-RC subunit III (PsaF) from Flaveria trinervia, the first psaF gene to be isolated from a C4 plant. The deduced amino acid sequence showed a high degree of similarity to the corresponding protein of spinach which is a C3 species. A region of 17 hydrophobic amino acids in the C-terminal part of the F. trinervia protein was found to be especially conserved in all PsaF proteins studied so far (cyanobacteria and Chlamydomonas).Abbreviations PSI-RC Photosystem I reaction center - cTPs chloroplast-targeted-proteins - chl chlorophyll - SDS sodium dodecyl sulfate