Cyanobacterial metallothionein gene expressed in Escherichia coli. Metal-binding properties of the expressed protein.

The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT). To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase. The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT). E. coli expressing this gene showed enhanced (ca. 3-fold) accumulation of Zn.     

Biosynthesis of terpenoids. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) from Plasmodium falciparum.

The putative catalytic domain of an open reading frame from Plasmodium falciparum with similarity to the ispF gene of Escherichia coli specifying 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase was expressed in a recombinant E. coli strain. The recombinant protein was purified to homogeneity and was found to catalyze the formation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate at a rate of 4.3 micromol x mg(-1) x min(-1). At lower rates, the recombinant protein catalyzes the formation of 2-phospho-2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate and the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol. Divalent metal ions such as magnesium or manganese are required for catalytic activity. The enzyme has a pH optimum at pH 7.0. Recombinant expression of the full-length open reading frame afforded insoluble protein that could not be folded in vitro. The enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis.     

Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography

Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.     

Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals

The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli. Upon induction, E. coli harboring this cDNA clone contained a protein species readily labelled by [35S]cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case. We show that expression of metallothionein endows resistance in E. coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.     

High-level expression of fully active yeast flavocytochrome b2 in Escherichia coli.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from the yeast Saccharomyces cerevisiae and three singly substituted mutant forms (F254, R349 and K376) have been expressed in the bacterium Escherichia coli. The enzyme expressed in E. coli contains the protohaem IX and flavin mononucleotide (FMN) prosthetic groups found in the enzyme isolated from yeast, has an electronic absorption spectrum identical with that of the yeast protein and an identical Mr value of 57,500 estimated by SDS/polyacrylamide-gel electrophoresis. N-Terminal amino-acid-sequence data indicate that the flavocytochrome b2 isolated from E. coli begins at position 6 (methionine) when compared with mature flavocytochrome b2 from yeast. The absence of the first five amino acid residues appears to have no effect on the enzyme-catalysed oxidation of L-lactate, since Km values for the yeast- and E. coli-expressed wild-type enzymes were identical within experimental error. The F254 mutant enzyme expressed in E. coli also showed kinetic parameters essentially the same as those found for the enzyme from yeast. The R349 and K376 mutant enzymes had no activity when expressed in either yeast or E. coli. The yield of flavocytochrome b2 from E. coli is estimated to be between 500- and 1000-fold more than from a similar wet weight of yeast (this high level of expression results in E. coli cells which are pink in colour). The increased yield has allowed us to verify the presence of FMN in the R349 mutant enzyme. The advantages of E. coli as an expression system for flavocytochrome b2 are discussed.     

The C-terminus of CaMKII is truncated when expressed in E. coli

The neuronal enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key molecule in biochemical events necessary for learning and memory. The alpha-subunit of CaMKII expressed in E. coli as well as in insect cells shows similar catalytic behavior [Praseeda, M., Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J. In Press]. The association domain of the enzyme has been crystallized in its native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product accompanies the full-length protein when expressed in E. coli. We show by epitope labeling and immunoblotting that the truncation occurs at the C-terminal half of the protein so that the N-terminal catalytic domain is complete in the truncated product. This supports the use of the preparation of alpha-CaMKII expressed in E. coli for studies on functions of the catalytic site. Our data will also be helpful in designing modified prokaryotic expression systems for CaMKII devoid of the trun-cation product, which are easier to use compared to the insect cell system.     

Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals.

5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group I422 (unit cell dimensions a = b = 130.7 A, c = 142.4 A). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (I422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.     

Cloning of the argF gene encoding the ornithine carbamoyltransferase from Corynebacterium glutamicum.

The argF gene encoding ornithine carbamoyl-transferase (OTCase; EC2.1.3.3) has been cloned from Corynebacterium glutamicum by transforming the Escherichia coli arginine auxotroph with the genomic DNA library. The cloned DNA also complements the E. coli argG mutant, suggesting a clustered organization of the genes in the genome. We have determined the DNA sequence of the minimal fragment complementing the E. coli argF mutant. The coding region of the cloned gene is 957 nucleotides long with a deduced molecular mass of about 35 kDa polypeptide. The enzyme activity and size of the expressed protein in the E. coli auxotroph carrying the argF gene revealed that the cloned gene indeed codes for OTCase. Analysis of the amino acid sequence of the predicted protein revealed a strong similarity to the corresponding protein of other bacteria.     

Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli.

We report on the cloning and characterization of the rfb gene cluster of the O75 lipopolysaccharide from a urinary tract isolate of Escherichia coli. Deletion cloning defined the minimum region of DNA that expressed the O75 antigen in E. coli host strains to be on a 12.4-kb insert. However, the E. coli strain expressing this region did not produce a polymerized O chain as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. A slightly larger DNA clone of 13.4 kb produced a polymerized O chain in E. coli S phi 874 but was found to be abnormal in its distribution over the surface membrane. Normal wild-type E. coli, as with Salmonella spp., has a bimodal distribution of the lipopolysaccharide on the surface which is seen as an abundance of long and short O chains attached to the lipid A-core structure. We found in a region adjacent to the cloned rfb region, and on the opposite side from where the putative polymerase (rfc) is encoded, a novel protein of 35.5 kDa expressed from a 1.75-kb DNA fragment. This protein was shown to complement in trans the E. coli strains carrying plasmids that expressed abnormal, unregulated lipopolysaccharides. The expression of these complemented strains was bimodal in distribution. Mutation of the gene encoding this protein destroyed its ability to regulate O-chain distribution. We propose to call this regulator gene rol, for regulator of O length.     

Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8.

The gene coding for the 100 kDa monomeric protein (P100) of the S layer of Thermus thermophilus HB8 has been cloned in the Escherichia coli plasmid vector pUC9. Recombinant plasmids were selected by colony screening with anti-P100 rabbit antiserum. The gene, named slpA (for surface layer protein A), was identified in a bacterial clone harboring a hybrid plasmid, pMF4, with a 5.8-kbp insert. This plasmid consistently expressed a protein specifically recognized by anti-P100 antiserum. Expression was apparently independent of Plac, indicating that the promoter for P100 is functional in E. coli. Most E. coli strains transformed with plasmids containing the 5.8-kbp insert cloned in pMF4 expressed two proteins with apparent masses of 52 and 50 kDa, which were strongly recognized by anti-P100 antiserum in Western immunoblots. The 52-kDa fragment could be overproduced, and the sequence of the N-terminal undecapeptide, determined by microsequencing, indicated that it could correspond to the N-terminal domain of P100. Expression of slpA in lon mutants of E. coli led to accumulation of a protein indistinguishable from native P100, indicating that the complete gene was cloned and that the product of lon, protease La, was involved in proteolytic degradation of P100 synthesized in E. coli.     

Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae. Purification and characterization

The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.     

Bacillus licheniformis6816碱性蛋白酶基因在E.coli中的克隆和表达

用PCR方法从地衣芽孢杆菌6816中扩增了碱性蛋白酶基因（apr),扩增的1.14kb的DNA片段插入到大肠杆菌载体pET-20b中,构建成重组分泌型表达载体pAPR1。pAPR1中碱性蛋白酶基因在大肠杆菌宿主JM109（DE3）中得到表达,SDS-PAGE分析显示融合表达产物的分子量为30kD,同核酸序列测定所推导的值相符,表达产物占细胞总蛋白的7.5%,重组菌的酶活比出发菌株提高了3.3倍,研究发现,重组的碱性蛋白酶在进入大肠杆菌周质空间时存在前肽自动脱落的现象。     

Bacterial IscU is a well folded and functional single domain protein.

Iron-sulfur clusters are widely represented in most organisms, but the mechanism of their formation is not fully understood. Of the two main proteins involved in cluster formation, NifS/IscS and NifU/IscU, only the former has been well studied from a structural point of view. Here we report an extensive structural characterization of Escherichia coli IscU. We show by a variety of physico-chemical techniques that E. coli IscU construct can be expressed to high purity as a monomeric protein, characterized by an alphabeta fold with high alpha-helix content. The high melting temperature and the reversibility of the thermal unfolding curve (as measured by CD spectroscopy) hint at a well ordered stable fold. The excellent dispersion of cross peaks in the (1)H-(15)N correlation spectrum is consistent with these observations. Monomeric E. coli IscU is able to provide a scaffold for Iron-sulfur cluster assembly, but has no direct interaction with either Fe(II) or Fe(III) ions, suggesting the need of further partners to achieve a stable interaction.     

Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a 4''-phosphopantetheine prosthetic group.

Rhizobium species produce a protein product of the nodF gene that has a limited but recognizable homology to the well-characterized acyl carrier protein (ACP) of Escherichia coli. NodF functions together with NodE in generating a host-specific response to the plant host in the interchange of signals leading to the effective nodulation of roots (H.P. Spaink, J. Weinman, M.A. Djordjevic, C.A. Wijffelman, R.J.H. Okker, and B. J.J. Lugtenberg, EMBO J. 8:2811-2818, 1989; B. Scheres, C. van de Wiel, A. Zalensky, B. Horvath, H. Spaink, H. van Eck, F. Zwartkruis, A.M. Wolters, T. Gloudemans, A. van Kammen, and T. Bisseling, Cell 60:281-294, 1990). The nodFE region of Rhizobium leguminosarum has been cloned into a multicopy plasmid and has been shown in R. leguminosarum to code for a flavonoid-inducible protein that is effectively labeled by radioactive beta-alanine added to the growth medium. After purification, the labeled protein migrates as a single band with an apparent molecular weight of 5,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, more rapidly than E. coli ACP. In contrast, in native gels the protein is resolved into two bands, both identified as NodF by analysis of the amino terminus and both migrating more slowly than E. coli ACP. Pulse-chase experiments with labeled beta-alanine suggested that the slower-moving band may be the precursor of the faster band. The NodF protein carries a 4'-phosphopantetheine as a prosthetic group. A NodF fusion protein under the control of the lac promoter is expressed in E. coli and is labeled with beta-alanine, indicating that it is recognized by the ACP synthase of E. coli. The ACP phosphodiesterase of E. coli, which catalyzes the release of phosphopantetheine from E. coli ACP, does not remove phosphopantetheine from NodF.     

Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.     

Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768

The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.     

Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.

The Escherichia coli AlkB protein is involved in protecting cells against mutation and cell death induced specifically by SN2-type alkylating agents such as methyl methanesulfonate (MMS). A human cDNA encoding a polypeptide homologous to E.coli AlkB was discovered by searching a database of expressed sequence tags (ESTs) derived from high throughput cDNA sequencing. The full-length human AlkB homolog (hABH) cDNA clone contains a 924 bp open reading frame encoding a 34 kDa protein which is 52% similar and 23% identical to E.coli AlkB. The hABH gene, which maps to chromosome 14q24, was ubiquitously expressed in 16 human tissues examined. When hABH was expressed in E.coli alkB mutant cells partial rescue of the cells from MMS-induced cell death occurred. Under the conditions used expression of hABH in skin fibroblasts was not regulated by treatment with MMS. Our findings show that the AlkB protein is structurally and functionally conserved from bacteria to human, but its regulation may have diverged during evolution.     

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.     

The glutamate uptake regulatory protein (Grp) of Zymomonas mobilis and its relation to the global regulator Lrp of Escherichia coli.

After being expressed in Escherichia coli JC5412, which is defective in glutamate transport, a Zymomonas mobilis gene which enabled this strain to grow on glutamate was cloned. This gene encodes a protein with 33% amino acid identity to the leucine-responsive regulatory protein (Lrp) of E. coli. Although overall glutamate uptake in E. coli was increased, the protein encoded by the cloned fragment repressed the secondary H+/glutamate transport system GltP by interaction with the promoter region of the gltP gene. It also repressed the secondary, H(+)-coupled glutamate uptake system of Z. mobilis, indicating that at least one role of this protein in Z. mobilis is to regulate glutamate transport. Consequently, it was designated Grp (for glutamate uptake regulatory protein). When expressed in E. coli, Grp repressed the secondary H+/glutamate transport system GltP by binding to the regulatory regions of the gltP gene. An lrp mutation in E. coli was complemented in trans with respect to the positive expression regulation of ilvIH (coding for acetohydroxy acid synthase III) by a plasmid which carries the grp gene. The expression of grp is autoregulated, and in Z. mobilis, it depends on growth conditions. The putative presence of a homolog of Grp in E. coli is discussed.     

Bioaccumulation of copper ions by Escherichia coli expressing vanabin genes from the vanadium-rich ascidian Ascidia sydneiensis samea

The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T. Ueki, T. Adachi, S. Kawano, M. Aoshima, N. Yamaguchi, K. Kanamori, and H. Michibata, Biochim. Biophys. Acta 1626:43-50, 2003). The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro. In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space. We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed. The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 micro M copper (II) ions were initially added. The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions. These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E. coli.