Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.     

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.     

Subtypes of the plasmid-encoded serine protease EspP in Shiga toxin-producing Escherichia coli: distribution, secretion, and proteolytic activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPalpha, espPbeta, espPgamma, and espPdelta), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPbeta, espPgamma, and espPdelta genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPalpha, espPbeta, espPgamma, and espPdelta in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPalpha (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPgamma cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPbeta and EspPdelta either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.     

Influence of hydrophobic mismatch on the catalytic activity of Escherichia coli GlpG rhomboid protease

Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. High-resolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10–12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.     

Periplasmic Secretion of Functional Ovotransferrin N-Lobe in Escherichia coli

The cytoplasmic and periplasmic production systems of ovotransferrin N-lobe in Escherichia coli were constructed. The periplasmic, but not cytoplasmic product, was found to have the ironbinding function.     

Expression of 15N-labeled eukaryotic cytochrome c in Escherichia coli

 We present a simple and inexpensive method for producing ¹⁵N-labeled Saccharomyces cerevisiae iso-1-cytochrome c in Escherichia coli. The labeled protein gives excellent NMR spectra. Received: 18 December 1998 / Accepted: 27 January 1999     

Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease.

The importance of proteases in gene regulation is well documented in both prokaryotic and eukaryotic systems. Here we describe the first example of genetic regulation controlled by the Escherichia coli Clp ATP-dependent serine protease. Virulent mutants of bacteriophage Mu, which carry a particular mutation in their repressor gene (vir mutation), successfully infect Mu lysogens and induce the resident Mu prophage. We show that the mutated repressors have an abnormally short half-life due to an increased susceptibility to Clp-dependent degradation. This susceptibility is communicated to the wild type repressor present in the same cell, which provides the Muvir phages with their trans-dominant phenotype. To our knowledge this is the first case where the instability of a mutant protein is shown to trigger the degradation of its wild type parent.     

Escherichia coli signal peptide peptidase A is a serine-lysine protease with a lysine recruited to the nonconserved amino-terminal domain in the S49 protease family

Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.     

Characterization of an alkaline serine protease from an alkaline-resistant Pseudomonas sp.: Cloning and expression of the protease gene in Escherichia coli

Summary A gene, aprP, encoding an extracellular alkaline serine protease from a newly isolated Pseudomonas sp. KFCC 10818 was cloned and characterized. Nucleotide sequence analysis revealed an open reading frame of 1,266 nucleotides which could encode a polypeptide comprised of 422 amino acids. The C-terminal 283 residues showed an overall sequence homology with the subtilisin-type serine proteases. When expressed in E. coli, the alkaline protease, AprP, was released to the culture medium. The purified AprP was most active at pH 11. The k_Cat/K_m value of this enzyme was 9.2 × 10³ S^–1mM^–1, which is much higher than those of subtilisins.     

Nucleotide sequences of two serine tRNAs with a GGA anticodon: the structure-function relationships in the serine family of E. coli tRNAs.

We have determined the nucleotide sequence of the major species of E. coli tRNASer and of a minor species having the same GGA anticodon. These two tRNAs should recognize the UCC and UCU codons, the most widely used codons for serine in the highly expressed genes of E. coli. The two sequences differ in only one position of the D-loop. Neither tRNA has a modified adenosine in the position 3'-adjacent to the anticodon. This can be rationalized on the basis of a structural constraint in the anticodon stem and may be related to optimization of the codon-anticodon interaction. Comparison of all E.coli serine tRNAs (and that encoded by bacteriophage T4) reveals characteristic (possibly functional) features. Evolutionary analysis suggests an eubacterial origin of the T4 tRNASer gene and the existence of a recent common ancestor for the tRNASerGGA and tRNASerGUC genes.     

Signal peptide digestion in Escherichia coli. Effect of protease inhibitors on hydrolysis of the cleaved signal peptide of the major outer-membrane lipoprotein

Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin-treated cell is processed to the mature form [Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712; (1982) J. Biol. Chem. 257, 5177-5182]. When this precursor-containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear. This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel; and (c) the results of labeling of the peptide with [3H]leucine, [35S]methionine and [3H]arginine were consistent with the amino acid composition of the signal peptide. The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named 'signal peptide peptidase' when the envelope fraction was washed to remove the inhibitors. The hydrolysis was inhibited by re-addition of these inhibitors. The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor.     

Characterization of the StcE protease activity of Escherichia coli O157:H7

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.     

Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli

Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.     

Effect of different positively charged amino acids, C-terminally of the signal peptidase cleavage site, on the translocation kinetics of a precursor protein in Escherichia coli K-12

Abstract Introduction of positively charged amino acids immediately downstream of the signal sequence in prokaryotic precursor proteins is known to affect the export process. However, it is not clear whether different positively charged amino acids affect the export process similarly. To investigate this, the glutamate at position +2 of outer membrane protein PhoE was substituted by arginine, lysine of histidine. Pulse-chase experiments revealed that the Lys and Arg residues at position +2 caused a reduced processing rate, and that the effect was markedly more severe in the case of the Arg residue. Trypsin accessibility experiments revealed that the accumulated precursors were present in the cytoplasm. Since the degree of the inhibitory effect corresponded to the p K _{r a} of the different positively charged amino acids, this suggests that the positively charged residues must be deprotonated during the secretory process.     

Leucine and serine induce mecillinam resistance in Escherichia coli.

Summary We have previously shown that resistance to the -lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and ReIA-dependent induction of the stringent response. We show here that our E. coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of lleucine or L-serine (or cysteine, which inactivates the antibiotic). The resistance, which is not a transient effect and does not depend on the physiological state of the cells when plated, is specific for mecillinam and is reversed by the presence of isoleucine and valine in the medium. At least in the case of serine, the resistance is ReIA-dependent. We conclude that the presence of leucine and serine in the growth medium cause partial starvation for isoleucine/valine, leading to induction of the stringent response and concomitant resistance to mecillinam.     

Biophysical studies of signal peptides: Implications for signal sequence functions and the involvement of lipid in protein export

This review discusses efforts to understand the mode of action of signal sequences by biophysical study of synthetic peptides corresponding to these protein localization signals. On the basis of reports from several laboratories, it is now clear that signal peptides may adopt a variety of conformations, depending on their local environment. In membrane-mimetic systems like detergent micelles or lipid vesicles, they have a high tendency to form helices. Ability to take up a helical conformation appears to be required at some point in the function of a signal sequence, since some peptides corresponding to export-defective signal sequences display reduced helical potential. By contrast, functional signal sequences share a high capacity to adopt helices. High affinity for organized lipid assemblies, like monolayers or vesicles, is also a property of functional signal sequences. This correlation suggests a role for direct interaction of signal sequences with the lipids of the cytoplasmic membranein vivo. Supporting this role are studies of the influence of signal peptides on lipid structure, which reveal an ability of these peptides to pertub lipid packing and to alter the phase state of the lipids. Insertion of the signal sequencein vivo could substantially reduce the barrier for translocation of the mature chain. Lastly, synthetic signal peptides have been added to native membranes and found to inhibit translocation of precursor proteins. This approach bridges the biophysical and the biochemical aspects of protein export and promises to shed light on the functional correlates of the properties and interactions observed in model systems.