An antibody-based affinity chromatography tool to assess Cu,Zn superoxide dismutase (SOD) G93A structural complexity in vivo

‘Conformational diseases’ are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1^G93A mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSOD^G93A rats provided evidence about heterodimers rSOD–hSOD^G93A formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSOD^G93A showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues.     

抗菌肽P7抑制大肠杆菌的非膜作用机制北大核心CSCD

【目的】研究抗菌肽P7抑制大肠杆菌的非膜作用机制。【方法】P7与溴化乙锭竞争结合大肠杆菌基因组DNA的荧光光谱,分析P7与DNA的结合方式;流式细胞术分析P7与大肠杆菌基因组DNA结合对细菌细胞周期的影响;采用磁珠富集和PCR扩增相结合的方法分析P7特异结合的DNA序列;通过实时荧光定量PCR分析P7对大肠杆菌DNA复制和SOS损伤修复基因表达的影响;核酸染料的荧光分析研究P7对大肠杆菌DNA和RNA合成的影响。【结果】P7以嵌插的方式作用于大肠杆菌基因组DNA碱基对并形成肽-DNA复合物,使溴化乙锭-DNA复合体系的荧光强度减弱。P7可以显著增加大肠杆菌细胞周期中S期细胞数目,抑制大肠杆菌DNA复制。P7特异性结合rnh A使该基因表达水平显著下调2.24倍。同时,在肽的影响下参与大肠杆菌DNA复制相关的ssb、dna G、lig B和rnh A基因的表达水平显著下调(P<0.05),DNA损伤修复的rec A和rec N基因显著上调(P<0.05)。P7可降低大肠杆菌DNA和RNA的合成。【结论】P7特异性地结合rnh A序列引起大肠杆菌DNA的损伤并抑制大肠杆菌的DNA复制。在P7的影响下,参与大肠杆菌DNA复制相关的基因的表达水平下调,DNA损伤修复基因显著上调,同时抑制大肠杆菌DNA和RNA的合成。     

Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression

To elucidate the effects of codon optimization and chaperone coexpression on the heterologous expression of mammalian cytochrome P450s (P450) in Escherichia coli, the expression of P450s 2B1, 2S1, 2U1, 2W1, and 27C1 were investigated. With codon optimization for N-terminus or the entire gene, the expression levels of P450 27C1, 2U1 and 2W1 increased 22-fold, 3.6-fold and 2.1-fold, respectively, while those for P450s 2B1 and 2S1 remained unchanged. With coexpression of E. coli molecular chaperones GroEL/ES, the expression level increased up to 14-fold for P450 27C1, and 3- to 5-fold for P450s 2B1, 2S1, and 2W1. Simultaneous application of these two techniques resulted in synergetic effects.     

Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli

Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli.     

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Inhibition of gene expression of T7-related phages by prophage P1

Summary The gene expression of nine phages of the T7 group was compared after infection of Escherichia coli B(P1). With the exception of phage 13a which grew normally, all of them infected E. coli B(P1) abortively. Differences were found in the efficiency of host killing which ranged from 100% for phage 13a to 37% for phage A1122. Infection by T7 prevented colony formation by about 70% of the cells but they showed filamentous growth until about 2h after infection. It was shown by SDS-polyacrylamide gel electrophoresis and autoradiography of [³⁵S]methionine-labelled phage-coded proteins that all phages except for 13a showed measurable expression only of the early genes. No correlation was observed between killing capacity and the pattern of gene expression, and the ability to hydrolyse S-adenosyl-methionine (SAM, a cofactor for the P1 restriction endonuclease) by means of a phage-coded SAMase. Mixed infection of E. coli B(P1) with 13a and T7 yielded mixed progeny indistinguishable from that observed after mixed infection of the normal host E. coli B. Genetic crosses with amber mutants of 13a and T7 showed that the 13a marker opo ⁺ (overcomes P one), required for growth on B(P1), is located in the early region, to the left of gene 1 (RNA polymerase gene).     

Identification,Molecular Cloning,and Sequence Analysis of a Deoxyribose Aldolase in <Emphasis Type="Italic">Streptococcus mutans</Emphasis> GS-5

Bacterial fitness in the environment, where nutrients are limited and competition is intense, plays a central role in survival and virulence of the organisms. Deoxyribose aldolase, found in several species of bacteria, is known to be involved in the catabolism of deoxynucleosides arising from dead cells, thereby giving a selective advantage to the microorganisms with a capability to consume DNA as an alternative carbon and energy source. A gene encoding a deoxyribose aldolase gene (deoC) was identified in the cariogenic Streptococcus mutans strain GS-5 by comparative sequence analysis and gene cloning. The gene encodes a protein of 220 amino acids, having a predicted molecular weight of 23.3 kDa with a pI of 5.44. The gene was cloned into the expression vector pFLAG-1, and the biological function of the gene product was analyzed by a complementation assay with a deoC^– Escherichia coli mutant S063. Transformation of the E. coli S063 with the plasmid construct allowed this organism to grow on glucose minimal medium supplemented with 2 mM deoxyadenosine or deoxythymidine. These results showed activity of deoxyribose aldolase, confirming the identity of the gene. Utilization of exogenous deoxynucleotides as a carbon and energy source may confer a survival and growth advantage to S. mutans over other bacteria in dental plaque, suggesting that deoxyribose aldolase may be a contributing factor to virulence.     

Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.

Summary The synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.Abbreviations and Symbols deoA (previously designated tpp) Genes coding for: thymidine, phosphorylase - deoB (previously designated drm) deoxyribomutase - deoC (previously designated dra) deoxyriboaldolase - deoD (previously designated pup) purine nucleoside phosphorylase - udp uridine phosphorylase - cytR regulatory gene for cdd, udp, deoC, deoA, deoB, and deoD - deoR (previously designated nucR) regulatory gene for deoC, deoA, deoB, and deoD Enzymes (EC 2.4.2.1) Purine nucleoside phosphorylase or purine nucleoside: orthophosphate(deoxy)ribosyltansferase - (EC 2.4.2.4) thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - (EC 2.4.2.3) uridine phosphorylase or uridine: orthophosphate ribosyltransferase - (EC 4.1.2.4) deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - (EC 2.7.5.6) phosphodeoxyribomutase The deo operon is defined as the gene cluster consisting of deoC deoA deoB deoD. The deo enzymes are the four enzymes encoded by the four genes of the deo operon. cAMP: cyclic adenosine 3,5-monophosphate. CRP: cyclic AMP receptor protein. dRib-5P: deoxyribose-5-phosphate. THUR: 3,4,5,6-tetrahydrouridine; EDTA: ethylene-diamine-tetra-acetate.     

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.     

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses.     

Expression, Purification, and Characterization of a Catalytically Active Human Cytochrome P450 1A2:Rat NADPH-Cytochrome P450 Reductase Fusion Protein

An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression inEscherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5′ end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3′ end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser–Thr. This fusion plasmid was expressed inE. coliand the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP–agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochromecreduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufinO-deethylation and phenacetinO-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase inE. colienhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.     

Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g_CDW⁻¹, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g_CDW⁻¹). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.     

Modification of Cysteine 111 in human Cu,Zn-superoxide dismutase

Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325 nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. In disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5′-dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.     

Outer membrane protein P1 is the CEACAM‐binding adhesin of Haemophilus influenzae

Haemophilus influenzae is a Gram‐negative pathogen colonizing the upper respiratory tract mucosa. H. influenzae is one of several human‐restricted bacteria, which bind to carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs) on the epithelium leading to bacterial uptake by the eukaryotic cells. Adhesion to CEACAMs is thought to be mediated by the H. influenzae outer membrane protein (OMP) P5. However, CEACAMs still bound to H. influenzae lacking OMP P5 expression, and soluble CEACAM receptor ectodomains failed to bind to OMP P5, when heterologously expressed in Escherichia coli. Screening of a panel of H. influenzae OMP mutants revealed that lack of OMP P1 completely abrogated CEACAM binding and supressed CEACAM‐mediated engulfment of H. influenzae by epithelial cells. Moreover, ectopic expression of OMP P1 in E. coli was sufficient to induce CEACAM binding and to promote attachment to and internalization into CEACAM‐expressing cells. Interestingly, OMP P1 selectively recognizes human CEACAMs, but not homologs from other mammals and this binding preference is preserved upon expression in E. coli. Together, our data identify OMP P1 as the bona fide CEACAM‐binding invasin of H. influenzae. This is the first report providing evidence for an involvement of the major OMP P1 of H. influenzae in pathogenesis.     

Regulatory mutants of the deo regulon in Salmonella typhimurium

Summary A method is described for isolating mutants which are constitutive for thymidine phosphorylase. The mutants isolated are also constitutive for all of the enzymes of the deo regulon and are unlinked to the deo genes suggesting that they have a defect in a regulatory gene. We have designated this regulatory gene deo R.     

Effect of chromosomal integration on catalytic performance of a multi-component P450 system in Escherichia coli

Cytochromes P450 are useful biocatalysts in synthetic chemistry and important bio-bricks in synthetic biology. Almost all bacterial P450s require separate redox partners for their activity, which are often expressed in recombinant Escherichia coli using multiple plasmids. However, the application of CRISPR/Cas recombineering facilitated chromosomal integration of heterologous genes which enables more stable and tunable expression of multi-component P450 systems for whole-cell biotransformations. Herein, we compared three E. coli strains W3110, JM109, and BL21(DE3) harboring three heterologous genes encoding a P450 and two redox partners either on plasmids or after chromosomal integration in two genomic loci. Both loci proved to be reliable and comparable for the model regio- and stereoselective two-step oxidation of (S)-ketamine. Furthermore, the CRISPR/Cas-assisted integration of the T7 RNA polymerase gene enabled an easy extension of T7 expression strains. Higher titers of soluble active P450 were achieved in E. coli harboring a single chromosomal copy of the P450 gene compared to E. coli carrying a medium copy pET plasmid. In addition, improved expression of both redox partners after chromosomal integration resulted in up to 80% higher (S)-ketamine conversion and more than fourfold increase in total turnover numbers.     

Overcoming difficulties on synthesis of cardiac troponin-I

Human cardiac troponin-I (cTnI) is one of the most sensitive and specific indicators, used in the diagnosis of myocardial infarction. To produce the protein efficiently, Escherichia coli and Pichia pastoris systems were used. Initial trials for the expression in E. coli were not successful, although different expression vectors with different promoters were tested. This led us to use P. pastoris for the expression. After several trials with two different expression strains of P. pastoris, it was concluded that P. pastoris was also not an optimal expression host for cTnI. Comprehensive analysis of the expression systems indicated that an efficient expression is only possible when the gene is optimized for expression in E. coli. For this purpose, the gene was optimized in-silico, but edited manually afterwards. It was synthesized and cloned into pQE-2 vector. Expression was performed using routine experimental conditions. Thus, cTnI could be efficiently expressed from the optimized gene in E. coli. The expression and purification were practical and may be used for commercial purposes since a total yield of 25µg highly pure protein per milliliter of culture could be obtained. The protein was in its ready-to-use form for many biological applications, including as a standard in diagnostic tests and an antigen for antibody production.     

15N-labeled P22 c2 repressor for nuclear magnetic resonance studies of protein-DNA interactions

The salmonella phage P22 c2 repressor was produced with 90% ¹⁵N isotope labeling of all leucines, using the expression system E. coli W3110 lac I ^Q/pTP 125. The N-terminal DNA-binding domain 1–76 was obtained by chymotrypsin cleavage. Its characterization by biochemical techniques, mass spectrometry, and one- and two-dimensional nuclear magnetic resonance (NMR) showed that highly residue-selective isotope labeling was achieved with the minimal growth medium used. The ability to obtain such isotope labeling opens new avenues for NMR studies of protein-DNA interactions in the P22 operator system.