Single Protein Production Research Articles

Enhanced Production of Functional Extracellular Single Chain Variable Fragment Against HIV-1 Matrix Protein from Escherichia coli by Sequential Simplex Optimization

The optimal culture condition for extracellular recombinant single chain variable fragment anti HIV-1 p17 protein (scFv anti-p17) production in Escherichia coli HB2151 was investigated by the sequential simplex optimization (SS) method. Five variable parameters were submitted in the fermentation process. The most favorable condition obtained from 19 independent experiments was as followed: 58 µM of IPTG induction to 1.7 OD600 nm at 25.5°C for 16 h with 202 rpm agitation rate. The amount of secreted scFv anti-p17 at the optimal condition was 38% higher than under the control condition. The binding activity of soluble extracellular scFv anti-p17 protein increased 95.5% and 73.2% in comparison with the control condition and non-optimized condition respectively. The soluble scFv anti-p17 from crude HB2151 lysated was subsequently purified by immobilized metal ion affinity chromatography (IMAC) with His-tag. The purified scFv anti-p17 was intact and retained its antigen-binding affinity against HIV-1 p17. We demonstrated that the sequential simplex optimization method was a key for exertion of high yield with fewer experimental requirements for acquiring of large scale secretory protein production.

Design and Selection of a Synthetic Operon

Cell-free systems are showing increasing promise for biosynthesis of both proteins and small molecules. However, in vitro transcription and translation reactions have so far primarily been used for the production of single proteins. In order to demonstrate the possibilities for coupled reactions, we designed synthetic operons that included different combinations of wild-type or evolved biotin ligases and streptavidins and demonstrated a mechanism for self-selection of operons following expression in vitro. Peptide substrates for biotin ligase were conjugated to the DNA operons and could be modified by a biotin ligase specific for either biotin or desthiobiotin and subsequently captured via a streptavidin specific for either biotin or desthiobiotin.

The Protective Effect of a Metallic Selenopeptide with Superoxide Dismutase and Glutathione Peroxidase Activities Against Alcohol Induced Injury in Hepatic L02 Cells

A 65-mer peptide (Se–CuZn–65P) is an enzyme mimic with antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX). The metallic selenopeptide imitate was obtained through a single protein production system and cysteine auxotrophic expression technique, and it enters cells in the assistance of the trans-activator of transcription peptide. Because Se–CuZn–65P was provided with the antioxidant activities of SOD and GPX, it can be a potential drug for the prevention of alcoholic liver diseases. Therefore, the protective effects of Se–CuZn–65P on ethanol-induced L02 cells were evaluated in vitro. Our results revealed that Se–CuZn–65P significantly protected the hepatocytes against ethanol-induced cell cytotoxicity which displayed by increase of cell viability, migration and decrease of reactive oxygen species (ROS), malondialdehyde (MDA), lactate dehydrogenase (LDH), aspartate aminotransferase (AST). Furthermore, the experiments of TUNEL assay, flow cytometry assay, expression levels determination of apoptosis-related gene (Bad, Bax, Bcl-xL, Bcl-2) and protein (pro-caspase3, PARP cleavage) indicated that Se–CuZn–65P inhibited ethanol-induced L02 cell apoptosis. Mechanically, Se–CuZn–65P could attenuate the oxidative damage of mitochondria in ethanol-induced L02 cells. Se–CuZn–65P prevented ethanol-triggered ROS and MDA generation in L02 cells and inhibited the release of the AST and LDH. Se–CuZn–65P also restored the equilibrium of proapoptotic and antiapoptotic genes including Bad, Bax, Bcl-xL, and Bcl-2. And the reduction of procaspase-3 and the cleavages of PARP were attenuated by Se–CuZn–65P. Thereby, the L02 cell apoptosis were significantly prevented by treatment with Se–CuZn–65P, mainly due to attenuation of oxidative stress. In summary, Se–CuZn–65P could prevent ethanol-induced liver injury through enhancing hepatocyte antioxidant abilities in intracellular.

High level soluble expression, purification, and characterization of human ciliary neuronotrophic factor in Escherichia coli by single protein production system

Ciliary neurotrophic factor (CNTF) is characterized as a neuropoietic cytokine for a broad spectrum of neurons, leading to its evaluation in humans suffering from neurodegenerative diseases. Due to its wide range of biological applications, high yield production of soluble biologically active recombinant human CNTF (rhCNTF) in heterologous expression system is demanded. Many attempts had been undertaken to product rhCNTF in Escherichia coli (E. coli), however, the expression level of rhCNTF was low and most of which formed insoluble inclusion bodies. In this study, we described a new and efficient method to express rhCNTF. The human CNTF gene was codon optimized and then expressed by the single protein production (SPP) expression system in E. coli. The results showed that rhCNTF was expressed as a soluble biologically active protein, and upon purification, the final yield was about 250mg/L in shake flask with a specific neuroprotective activity in Aβ-induced SH-SY5Y cell injury model. Our study might open up a new strategy for large-scale production of functional rhCNTF for clinical applications as well as basic research.

Can Recombinant Human Glutathione Peroxidase 1 with High Activity Be Efficiently Produced inEscherichia coli?

Cellular glutathione peroxidase 1 (GPx1) is an important antioxidant selenoenzyme. Due to the presence of selenocysteine (Sec) encoded by UGA, normally recognized as a stop codon, the expression of mammalian GPx with traditional recombinant DNA technology is extremely difficult. In this study, a series of human GPx1 (hGPx1) mutants with significantly high catalytic activities were produced for the first time in an Escherichia coli BL21(DE3)cys auxotrophic strain using the single-protein production system. Cys residues in hGPx1 were mutated to Ser in turn because untargeted substitution of Sec in place of Cys resulted in the decline of recombinant selenoenzyme activity. The results of this work showed that the catalytic activities of the mutants increased progressively with decreasing number of noncatalytic Sec residues. Seleno-hGPx1-C2/78/115/156/202S with all Cys residues changed to Ser showed the highest activity (21,268 U/μmol), which was more than 10-fold higher than bovine liver GPx. This increase could be explained by structural analysis of hGPx1 mutants based on homology modeling and binding site analysis. These results lead to the hypothesis that the conversion of noncatalytic Sec residues to Ser may optimize the structure of seleno-GPx in this expression system and consequently increase the catalytic efficiency.

A Triple-Transgenic Immunotolerant Mouse Model

Avoiding unwanted immunogenicity is of key importance in the development of therapeutic drug proteins. Animal models are of less predictive value because most of the drug proteins are recognized as foreign proteins. However, different methods have been developed to obtain immunotolerant animal models. So far, the immunotolerant animal models have been developed to assess one protein at a time and are not suitable for the assessment of combination products. Our aim was to develop an animal model for evaluating the impact of manufacturing and formulation changes on immunogenicity, suitable for both single protein and combination products. We constructed two lines of transgenic mice expressing the three human coagulation factors, II, VII, and X, by inserting a single vector containing the three coagulation factors encoding sequences separated by insulator sequences derived from the chicken beta-globin locus into the mouse genome. Immunization of transgenic mice from the two lines and their wild-type littermates showed that transgenic mice from both lines were immunotolerant to the expressed human coagulation factors. We conclude that transgenic mice immunotolerant to multiple proteins can be obtained, and that these mice are potentially useful as animal models in the assessment of immunogenicity in response to manufacturing changes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1116–1124, 2013

Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.

Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression

BackgroundHistone methyltransferase enhancer of zeste homologue 2 (EZH2) forms an obligate repressive complex with suppressor of zeste 12 and embryonic ectoderm development, which is thought, along with EZH1, to be primarily responsible for mediating Polycomb-dependent gene silencing. Polycomb-mediated repression influences gene expression across the entire gamut of biological processes, including development, differentiation and cellular proliferation. Deregulation of EZH2 expression is implicated in numerous complex human diseases. To date, most EZH2-mediated function has been primarily ascribed to a single protein product of the EZH2 locus.ResultsWe report that the EZH2 locus undergoes alternative splicing to yield at least two structurally and functionally distinct EZH2 methyltransferases. The longest protein encoded by this locus is the conventional enzyme, which we refer to as EZH2α, whereas EZH2β, characterized here, represents a novel isoform. We find that EZH2β localizes to the cell nucleus, complexes with embryonic ectoderm development and suppressor of zeste 12, trimethylates histone 3 at lysine 27, and mediates silencing of target promoters. At the cell biological level, we find that increased EZH2β induces cell proliferation, demonstrating that this protein is functional in the regulation of processes previously attributed to EZH2α. Biochemically, through the use of genome-wide expression profiling, we demonstrate that EZH2β governs a pattern of gene repression that is often ontologically redundant from that of EZH2α, but also divergent for a wide variety of specific target genes.ConclusionsCombined, these results demonstrate that an expanded repertoire of EZH2 writers can modulate histone code instruction during histone 3 lysine 27-mediated gene silencing. These data support the notion that the regulation of EZH2-mediated gene silencing is more complex than previously anticipated and should guide the design and interpretation of future studies aimed at understanding the biochemical and biological roles of this important family of epigenomic regulators.

Sally Smith Hughes, Genentech: The Beginnings of Biotech

Drawing extensively on oral histories, Sally Smith Hughes, in her fascinating book, vividly recounts the blood, sweat and tears of the early days of ‘genetic engineers’ working at the bench, designing new biomolecules and capitalising their promises on Wall Street. This not only made for Genentech's success, but also revolutionised drug research and development. In concentrating on molecular biology (DNA and proteins), rather than on traditional chemistry, the genetic engineers succeeded in providing an entire apothecary of new biosynthetic drugs from natural substances within the body. The cloning and production of what would become a blockbuster drug, interferon, was exemplary of this spectacular endeavour. Through the lens of Genentech's foundering, one of the first and most successful genetic boutiques, Hughes traces back the mind-capturing pioneering efforts to copy or ‘clone’ the genes of higher organisms in bacteria by means of recombinant-DNA techniques and induce the bacteria to express them as proteins. The Cohen and Boyer experiments, which became a citation classic, demonstrated dramatically the potential impact of DNA recombinant engineering not only to fellow scientists, but also to a wider audience. Boyer's remark during the June 1973 Gordon conference that his experiment (together with Cohen) had created a novel combination of antibiotic resistant genes triggered immediate concern. Combined with the news that they had filed a patent application, this planted the seeds for what came to be known as the recombinant-DNA controversy. Hughes masterfully describes how both Cohen and Boyer individually pursued efforts to patent and explore the practical application of the recombinant-DNA technology during the heyday of the public DNA furore. The fundamental technology was still raw and industrially untested, with not a single protein product on the near horizon. Thus, the leading pharmaceutical and chemical corporations had difficulty in assessing the potential of what was regarded as a high-risk research area. Company executives just did not envisage the promising horizon that was so self-evident to Boyer in particular. Wait and see was not an option for Boyer, and together with the venture capitalist Bob Swanson, he took a calculated risk to start the genetic engineering firm ‘Genentech’. Obtaining exclusive licence rights to make recombinant pharmaceuticals and generating new research patents was regarded as critical to the firm's survival. This indeed proved to be vital in their early endurance as a supplier of technology to established corporations like Eli Lilly, and the business's subsequent exponential growth as a fully integrated and self-sufficient pharmaceutical company, allowing Genentech to continue as an independent, publicly traded firm until the twenty-first century.

Generation of selenoprotein with glutathione peroxidase activity by chemical modification of the single-chain variable fragment expressed in a single-protein production system and its antioxidant ability

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. Single-chain variable fragment (scFv) can be converted into seleniumcontaining single-chain variable fragment (Se-scFv) by chemical modification of the hydroxyl groups in scFv, thus Se-scFv possesses GPX activity and becomes a prodrug. To improve the expression of scFv and simplify its purification steps, Single-protein production (SPP) system was used to express scFv and chemical modification was used to synthesize Se-scFv. Therefore, we must construct a new scFv-WCD1-lessACA gene, which can express its mRNA not containing any ACA sequences and express its amino acid sequence of target protein (scFv) being same to scFv-WCD1. In this way, the scFv-WCD1-lessACA can be only expressed in SPP system and no other background proteins in the cells could be expressed. The expression results showed that high level of scFv-WCD1-lessACA synthesis was at least sustained for 96 h in the virtual absence of background protein synthesis. Then, selenocysteine (Sec) was incorporated into the scFv-WCD1-lessACA by chemical modification and resulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics of Se-scFv-WCD1-lessACA were determined. GPX activity was 2,563 U/μmol, its binding constant for GSH was 0.687 ×105/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe2+ (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS).

An Assay Suitable for High Throughput Screening of Anti-Influenza Drugs

We developed a novel drug screening system for anti-influenza A virus by targeting the M2 proton channel. In the SPP (Single Protein Production) system, E. coli cell growth occurs only in the presence of effective M2 channel inhibitors, and thus simple measurement of cell growth was used as readouts for drug screening. Two potential inhibitors for M2 (V27A) mutant were verified using this method, which inhibit both the mutant and wild-type M2 channels.

Searching for Alternatively Spliced Variants of Phospholipase Domain-Containing 2 (Pnpla2), a Novel Gene in the Retina

Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length PNPLA2 cDNA or a PNPLA2 cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots. PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.

Creation of a New protein by substituting all arginine residues by its toxic analogue, canavanine

Complete substitution of a specific amino acid in a protein with its analogue may create a novel protein with unprecedented properties but is quite challenging, particularly if the analogue is highly toxic to living cells. Canavanine (Can), an analogue of Arginine (Arg) found in certain leguminous plants is highly toxic, in which a ε‐methylene group in Arg is replaced with an oxa group, lowering the pKa value from 12.48 to 7.04. Here, we demonstrate for the first time the complete replacement of Arg residues in a protein with Can, as all the seven Arg residues in an mRNA interferase from Bacillus subtilis, MazF‐bs were replaced with Can. To achieve this, the ACA‐less gene was engineered to produce only MazF‐bs in the presence of Can in a condensed culture of the Single‐Protein Production (SPP) system using an E. coli Arg‐auxotroph strain. We show that all Arg residues were completely replaced with Can as judged by mass spectroscopy. The CD spectrum of MazF‐bs(Can) was almost identical to that of the wild‐type MazF‐bs(Arg) but MazF‐bs(Can) was less thermo‐stable than MazF‐bs(Arg). Interestingly, MazF‐bs(Can) cleaved RNA not only at the original UUACA cleavage sites, but also at the CCACU sites. This is the first report to show that the complete replacement of Arginine with Canavanine in a protein, resulting in acquiring of a new function.

Structural disorder within paramyxovirus nucleoproteins and phosphoproteins

This review focuses on the experimental data showing the abundance of structural disorder within the nucleoprotein (N) and phosphoprotein (P) from three paramyxoviruses, namely Nipah (NiV), Hendra (HeV) and measles (MeV) viruses. We provide a detailed description of the molecular mechanisms governing the disorder-to-order transition of the intrinsically disordered C-terminal domains (N(TAIL)) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins. We also show that a significant flexibility persists within N(TAIL)-XD complexes, which therefore provide illustrative examples of "fuzziness". The functional implications of structural disorder are discussed in light of the ability of disordered regions to establish a complex molecular partnership, thereby leading to a variety of biological effects. Taking into account the promiscuity that typifies disordered regions, we propose that the main functional advantage of the abundance of disorder within viruses would reside in pleiotropy and genetic compaction, where a single gene would encode a single (regulatory) protein product able to establish multiple interactions via its disordered regions, and hence to exert multiple concomitant biological effects.

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.

Characterization of PROFILIN genes from allotetraploid (Gossypium hirsutum) cotton and its diploid progenitors and expression analysis in cotton genotypes differing in fiber characteristics

The actin-binding protein profilin (PRF) plays an important role in cell growth and expansion by regulating the organization of the actin filaments. Recent studies have reported association between fiber elongation in cultivated cotton (Gossypium hirsutum) and PRF expression. In the present study, we cloned four genomic clones from allotetraploid cotton (G. hirsutum) and its putative diploid progenitors (G. arboreum and G. raimondii) designated GhPRF1_A, GhPRF1_D, GaPRF1, and GrPRF1 encoding cotton PRF and characterized their genomic structure, phylogenetic relationships and promoter structure. Sequence analysis of the coding regions of all clones resulted in a single protein product which revealed more than 80% similarity to most plant PRFs and a typical organization with an actin-binding and a polybasic phospholipid binding motif at the carboxy terminus. DNA blot hybridization suggested that PRF gene is present with more than one copy in the allotetraploid species G. hirsutum. Expression analysis performed in various organs of cultivated cotton revealed that the PRF gene was preferentially expressed in cotton fibers. Very low levels of expression were observed in whole flowers, while PRF transcripts were not detected in other organs examined. Furthermore, higher levels of expression were observed at the early stages of cotton fiber development (at 10days post anthesis), indicative that this gene may play a major role in the early stages of cotton fiber development. Quantitation of the expression by real-time PCR revealed higher expression levels in a G. hirsutum variety with higher fiber percentage compared to a variety with lower percentage. In addition, higher levels of expression were found in cultivated allotetraploid G. barbadense cotton species with higher fiber length in comparison to cultivated allotetraploid G. hirsutum.

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that (13)C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that (13)C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

Secretion of human superoxide dismutase in Escherichia coli using the condensed single‐protein‐production system

A secretion vector, pColdV for the Single-Protein-Production (SPP) system was constructed using the E. coli OmpA signal peptide. Using this vector, human superoxide dismutase (hSOD) was co-expressed with MazF, an ACA-specific mRNA interferase, allowing E. coli cells to produce only hSOD, which was secreted into the periplasmic space with a yield of ∼20% of total cellular proteins. The signal peptide was properly cleaved. Using cells overproducing DsbA protein, two S-S bridges were also properly formed to yield enzymatically active SOD. A well resolved heteronuclear single quantum coherence (HSQC) spectrum of hSOD isotope-labeled in the condensed SPP (cSPP) system was obtained by simply isolating the periplasmic fraction. These results indicate that human secretory proteins can be expressed well in the cSPP system using pColdV.

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

The Use of the Condensed Single Protein Production System for Isotope-Labeled Outer Membrane Proteins, OmpA and OmpX in E. coli

Gram-negative bacteria consist of two independent membranes, the inner cytoplasmic membrane and the outer membrane. The outer membrane contains a number of β-barrel proteins such as OmpF, OmpC, OmpA, and OmpX. In this article, we explored to use the condensed Single Protein Production (cSPP) system for isotope labelling of OmpA and OmpX for NMR structural study, both of which are known to consist of eight β-strands forming a barrel in the outer membrane. Using a deletion strain lacking all major outer membrane proteins, both OmpA and OmpX were expressed well in a 20-fold cSPP system. We demonstrated that outer membrane fractions prepared from the cSPP system in M9 medium containing ¹⁵N-NH₄Cl can be directly used for NMR structural study of the outer mebrane proteins without any further purification to get excellent [¹H-¹⁵N]-TROSY spectra. This method would be quite valuable for the study of pure proteins in their native membrane environment without the need of purification and reconstitution.