The Escherichia coli ATP-binding cassette (ABC) proteins

The recent completion of the Escherichia coli genome sequence ( Blattner et al ., 1997 ) has permitted an analysis of the complement of genomically encoded ATP-binding cassette (ABC) proteins. A total of 79 ABC proteins makes this the largest paralogous family of proteins in E . coli . These 79 proteins include 97 ABC domains (as some proteins include more than one ABC domain) and are components of 69 independent functional systems (as many systems involve more than one ABC domain). The ABC domains are often, but not exclusively, the energy-generating domains of multicomponent membrane-bound transporters. Thus, 57 of the 69 systems are ABC transporters, of which 44 are periplasmic-binding protein-dependent uptake systems and 13 are presumed exporters. The genes encoding these ABC transporters occupy almost 5% of the genome. Of the 12 systems that are not obviously transport related, the function of only one, the excision repair protein UvrA, is known. A phylogenetic analysis suggests that the majority of ABC proteins can be assigned to 10 subfamilies. Together with statistical and, importantly, biological evidence, this analysis provides insight into the evolution and function of the ABC proteins.     

The metD D-methionine transporter locus of Escherichia coli is an ABC transporter gene cluster

The metD D-methionine transporter locus of Escherichia coli was identified as the abc-yaeE-yaeC cluster (now renamed metNIQ genes). The abc open reading frame is preceded by tandem MET boxes bracketed by the -10 and -35 boxes of a promoter. The expression driven by this promoter is controlled by the MetJ repressor and the level of methionine.     

Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.     

Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA(218)) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 x 10(4) E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23 degrees C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA(218) fusion protein at 23 and 30 degrees C but unexpectedly not at 37 degrees C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA(218) was detected only in the L448F mutant (AE104F) at 23 degrees C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA(218) fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37 degrees C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.     

Enhancement of the efficiency of secretion of heterologous lipase in Escherichia coli by directed evolution of the ABC transporter system

The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.     

Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)

The effect of two Escherichia coli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).     

Export and processing of MalE-LacZ hybrid proteins in Escherichia coli

Five classes of MalE-LacZ hybrid proteins have previously been characterized. These proteins differ in the amount of the maltose-binding protein (MBP) that is attached to beta-galactosidase. Although none of these proteins is secreted into the periplasm, the four larger classes of hybrid proteins, those that include an intact MBP signal peptide, are inserted into the cytoplasmic membrane, suggesting that the secretion process has at least been initiated. In this study, we demonstrated that some portion of the four larger hybrid proteins can be translocated across the cytoplasmic membrane, thus permitting processing of the signal peptide. We have found that hybrid proteins that include only a small portion of the mature MBP are inefficiently recognized as exported proteins, and translocation and processing of these appear to be relatively slow, posttranslational events. In marked contrast, hybrid proteins that include a substantial portion of the mature MBP are efficiently recognized, and translocation and processing of these occur very rapidly, possibly cotranslationally. Our results complement other studies and very strongly suggest a role for the mature MBP in the export process.     

Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli

Recombinant Escherichia colistrains harboring the genes from Alcaligenes eutrophusfor polyhydroxyalkanoate biosyn-thesis were constructed and compared for their ability to synthesize poly(3-hydroxybutyrate) in a defined medium with whey as the sole carbon source. The highest PHB concentration and PHB content obtained were 5.2 g/L and 81% of dry cell weight, respectively.     

GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli

Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-alpha2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use.     

The Escherichia coli metD locus encodes an ABC transporter which includes Abc (MetN), YaeE (MetI), and YaeC (MetQ)

We report that the genes abc, yaeC, and yaeE comprise metD, an Escherichia coli locus encoding a DL-methionine uptake system. MetD is an ABC transporter with Abc the ATPase, YaeE the permease, and YaeC the likely substrate binding protein. Expression of these genes is regulated by L-methionine and MetJ, a common repressor of the methionine regulon. We propose to rename abc, yaeE, and yaeC as metN, metI, and metQ, respectively.     

N-terminal acetylation of ectopic recombinant proteins in Escherichia coli

N-terminal acetylation is a protein modification common in eukaryotes, but rare in prokaryotes. Here, we characterized five mammalian stathmin-like subdomains expressed in Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoESI Q-TOF tandem mass spectrometry. We revealed that RB3(SLD) and RB3'(SLD) are N(alpha)-acetylated, whereas SCG10(SLD) and SCLIP(SLD), although identical up to residue 6, are not, as well as stathmin. To assess the influence of the N-terminal sequences on N(alpha)-acetylation, we exchanged residues 7 and 8 between acetylated RB3(SLD) and unacetylated SCG10(SLD), and showed that it reversed the acetylation pattern. Our results demonstrate that ectopic recombinant proteins can be extensively N(alpha)-acetylated in E. coli, and that the rules governing N(alpha)-acetylation are complex and involve the N-terminal region, as in eukaryotes.     

Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.     

Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli

Abstract

The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.     

Process conditions affecting proteolysis of recombinant proteins in Escherichia coli

The effects of process conditions on proteolysis of three recombinant IgG-binding proteins ZT3, ZZT3 and protein A in E. coli were studied. SDS/PAGE shows that the relative amount of degradation intermediates of ZT3 in a shake flask cultivation is much higher than that in a bioreactor culture. The rate of proteolysis of ZZT3 and protein A was also higher in shake flask cultures than in bioreactor cultures. The proteolysis rate constant of ZZT3 was 12/h in a shake flask but only 2.1/h in a bioreactor. Corresponding values for protein A were 2.4/h and 1.0/h, respectively. High proteolysis rate constants correlated with lower product yields.     

Expression of hepatitis A virus recombinant proteins in Escherichia coli

On the basis of coding regions on the fragments of genes P1 and P2 of hepatitis A virus (HAV) recombinant proteins of this virus have been synthesized in the prokaryotic expressing system of E. coli, isolated and studied with the use of sera obtained from hepatitis A patients. The capacity of HAV recombinant proteins for binding with the sera of patients with hepatitis A in the acute stage has been shown with the use of immunoblotting and the indirect solid-phase enzyme immunoassay. The results obtained in this investigation are discussed in the light of the possible use of recombinant proteins for the detection of HAV markers.     

Substrate recognition mechanism of the peptidase domain of the quorum-sensing-signal-producing ABC transporter ComA from Streptococcus

ComA of Streptococcus is a member of the bacteriocin-associated ABC transporters, which is responsible for both the processing of the propeptide ComC and secretion of the mature quorum-sensing signal. The quorum-sensing system is a bacterial intercellular communication system implicated in various functions including biofilm formation. In this study, the peptidase domains (PEPs) of the ComAs from six species of Streptococcus and ComCs from four species were expressed, purified, and characterized to address the mechanism of the substrate recognition of PEP. PEPs specifically cleaved ComCs after the Gly-Gly site in all the PEP-ComC combinations examined. The N-terminal leader region of ComC was found to form an amphiphilic alpha-helix structure upon binding to the PEP. Furthermore, mutagenesis studies revealed that four conserved hydrophobic residues in this leader region of ComC extending from -15 to -4 positions are critical in the interaction with PEP. Together with the double glycine motif, these structural features of ComC would explain the strict substrate specificity of the PEP.     

Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins

For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25 degrees C and 30 degrees C). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations.     

Assessment of iron toxicity using a luminescent bacterial test with an Escherichia coli recombinant strain

The toxic effect of the Fe²⁺ and Fe³⁺ ions on the luminescent recombinant Escherichia coli strain with the luxCDABE operon was studied in short- and long-term experiments. At 30-min exposure of bacteria to the iron ions, the effective concentrations of Fe²⁺ and Fe³⁺ resulting in acute toxicity (EC₅₀) were 8.5 and 1.3 mg/L, respectively. In the long-term (24 h) experiment, during active bacterial growth, the toxicity index for Fe²⁺ and Fe³⁺ was 65.5 and 62.8, respectively. Addition of the iron ions into the medium did not suppress growth, although it inhibited luminescence. Comparative analysis of the short- and long-term experiments made it possible to assess iron toxicity at the concentrations from 0.5 to 20 mg/L (as calculated for the Fe²⁺ and Fe³⁺ ions). Iron ions were found to affect only the reactions that were not vitally important for the cell. At the same time, they had no negative effect on the genetic mechanisms and protein synthesis, thus indicating non-specific toxicity of Fe²⁺ and Fe³⁺.