Expression Level Of Recombinant Protein Research Articles

High-level expression of codon-optimized Taq DNA Polymerase under the control of rhaBAD promoter

A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the L-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.

A flexible ‘plug and play’ bicistronic construct and its application in the screening of protein expression system in Escherichia coli

The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.

Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37°C and PDI/XBP-1s/37°C, the Adalimumab volume yield of PDI/XBP-1s/33°C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

Additivities for Soluble Recombinant Protein Expression in Cytoplasm of Escherichia coli

Recombinant protein expression in Escherichia coli is a fundamental technique in molecular biology and biotechnology. This review provides a comprehensive overview of various additivities to enhance the expression levels of soluble recombinant proteins in E. coli. The discussion encompasses five key aspects. Inducer Optimization: strategies for optimizing the inducer concentration to enhance protein expression. Autoinduction system optimization: the examination of glucose, lactose, and glycerol optimization within autoinduction systems to improve protein production. Osmolytes and osmoprotectants: an analysis of the use of osmolytes and osmoprotectants, such as sorbitol and glycine-betaine, to overcome with ease osmotic stress and enhance protein solubility. Ethanol additives: the impact of ethanol on E. coli physiology and its potential to improve recombinant protein expression. Cofactors and metabolic precursors: insights into the addition of cofactors, such as pyridoxal phosphate, riboflavin, thiamine, and pyridoxine, and the utilization of metabolic precursors to enhance the corresponding protein expression. This review highlights both the successful strategies and challenges in recombinant protein expression and provides insights into potential future research directions. Understanding and optimizing these factors is crucial for the efficient production of recombinant proteins for various applications in biotechnology. Furthermore, based on the analyzed data, we propose a straightforward scheme to optimize the additives in the cultivation medium.

Recombinant Myostatin: Isolation and Use in Vaccines

Protein myostatin is not only a potential target for therapeutic effects in pathologies of the muscular system, but also a tool for biotechnological regulation of the meat productivity of farm animals. The action of myostatin can be blocked by inducing autoantibody synthesis using recombinant myostatin (rMST) as an antigen. E. coli producer strain Bl21/pET28-MSTN was obtained with a high level of recombinant protein expression in inclusion bodies. Purified rMST was used to immunize sheep and rabbits. Using the created ELISA test system for the detection of antibodies to myostatin in the blood of sheep, it was shown that rMST is immunogenic (the working titer of sera is 1/400–1/800). As a result of vaccination of rabbits over a monthly period, the average body weight and average daily weight gain in animals of the experimental groups was observed to increase compared to the control group.

Efficient production and characterization of soluble active human β-1,2-N-acetylglucosaminyltransferase II in bacteria

In humans, almost all the cell surface and secreted glycoproteins are modified with complex-type N-glycans. Thus, it is essential to obtain complex-type N-glycans to fully understand the biological properties of glycoproteins. Here, human β-1,2-N-acetylglucosaminyltransferase II (hGnT-II), a Golgi-localized enzyme integral to complex-type N-glycan biosynthesis, was cloned as a truncated transmembrane form (GnT-II-ΔTM) and heterologously overexpressed in Escherichia coli. Our results showed that hGnT-II could be overexpressed in its soluble form by fusing the truncated enzyme with a thioredoxin (Trx)-tag in the Rosetta-Gami 2 strain. Using the optimized induction conditions, the expression level of recombinant protein was enhanced to yield approximately 4mg per liter culture after affinity purification. The enzyme exhibited appropriate glycosyltransferase activity, and the calculated Km value was 52.4μM, similar to the protein expressed in mammalian cells. Furthermore, the effect of MGAT2-CDG mutations on enzyme activity was also measured. These results suggested that the E.coli expression system was capable of the large-scale production of bioactive hGnT-II, which can be used for functional study and effective synthesis of complex-type N-glycans.

A histidine-rich fusion tag enables real-time monitoring of recombinant protein expression by Pauly reaction-based colorimetric assay

Commercially available recombinant expression systems always use fusion tags to facilitate target protein purification and SDS-PAGE analysis followed by Coomassie Brilliant Blue (CBB) staining is the classical method to validate the expression level of target protein, which is time-consuming, although not very laborious. Previously, we found that a histidine-rich elastin-like polypeptide (HRELP) tag could make its fusion proteins being quickly and specifically stained with Pauly's reagent. In this study, we designed a Pauly reaction-based colorimetric assay to real-time monitoring of the expression level of recombinant protein tagged HRELP and found that the absorption value of post-induction E. coli cells stained with Pauly's reagent correlated well with both the band intensity of the target protein from Pauly's reagent-stained and CBB-stained gels. Moreover, we found the colorimetric assay could also be helpful to roughly estimate the expression efficiency by using a poly-histidine-tagged protein, which has only 1.17% histidine residue. In our opinion, Pauly reaction-based colorimetric assay could significantly shorten the time to validate the over-expression of recombinant protein tagged with either HRELP or poly-histidine. And HRELP seemed to be an ideal fusion tag for it can not only facilitate protein purification but also simplify protein detection.

Characterization of a novel thermostable alkaline lipase derived from a compost metagenomic library and its potential application in the detergent industry.

Using composted soil samples, a metagenomic library consisting of 36,000 clones was constructed. Then, a novel lipase, Lip54q, which belongs to the VIII family of lipolytic enzymes, was identified from the metagenomic library by functional screening. To explore the enzymatic properties of Lip54q, lip54q was heterologous expressed in Escherichia coli with a high expression level of recombinant protein up to 720 mg/L. The recombinant enzyme showed the highest activity (28,160 U/mg) against a C10 substrate at pH 9.0 and 47°C, and was stable at temperatures ≤50°C and pH 8.0-11.0. Of particular interest, the surfactants, Tween-20, Tween-80 and Tritonx-100, exhibited strong promoting effects on Lip54q activities regardless of whether low concentrations (0.1%) or high concentrations (10%) were used. Application studies of Lip54q using six commercial detergents indicated that the enzyme had strong tolerance and immersion resistance to all six detergents. The results of oil-stain removal experiments suggested that addition of the enzyme to various commercial detergents could significantly improve the abilities of these detergents to remove oil-stains. Furthermore, the results of a molecular docking analysis of Lip54q showed that both the C10 substrate and linoleic acid molecules could form hydrogen bond interactions with the catalytic amino acids, Ser-268, Glu-168, and Asp-192, in the catalytic center of the enzyme, and the hydrogen bond distances were shorter. The electrostatic attraction between the enzyme and the substrate formed by the hydrogen bond with a shorter distance is stronger, which is conducive to the formation of a more stable complex between the enzyme and the substrate, thus increasing the activity of the enzyme to such substrate. These results 1ay a good foundation for application of this enzyme in the detergent industry in the future.

Protection against cystic echinococcosis in sheep using an Escherichia coli-expressed recombinant antigen (EG95) as a bacterin.

The EG95 recombinant vaccine is protective against cystic echinococcus in animal intermediate hosts. Preparation of the existing, registered EG95 vaccines involves semi-purification of the vaccine protein, adding to the cost of production. Truncation of the EG95 cDNA, shortening both the amino and carboxy-termini of the protein, leads to high levels of recombinant protein expression. The recombinant EG95 protein was prepared as a bacterin from clarified, whole bacterial lysate, and used in a vaccine trial in sheep against an experimental challenge infection with Echinococcus granulosus eggs. The EG95 bacterin was found to induce 98% protection. Use of this in a new generation EG95 vaccine would simplify production, facilitate new sources of the vaccine and potentially enhance uptake of vaccination in control of E. granulosus transmission.

Homologous recombination risk in baculovirus expression vector system

The baculovirus expression vector system (BEVS) is widely used for producing recombinant proteins. To achieve high expression level of recombinant proteins, baculoviral elements, such as enhancers, promoters, signal peptide coding sequences and 3′-UTR, have been extensively employed. There is a recombination risk derived from homologous sequences between viral genome and functional baculovirus-derived elements associated with foreign genes. Although homologous recombination have distinct biological functions, these potential adverse recombination may trigger a DNA fragment being inverted or looped out, resulting in the production of defective viruses and eventual yields declines of recombinant proteins. However, the risk of such homologous recombination has not been systematically assessed. Here, we measured the recombination rate using a promoter-less fluorescent reporter integrated with various lengths homologous of p10 coding region. Homologous fragments longer than 60 bp possess sufficient recombination probability and exerts effect on purity and integrity of virus. Shortening the length of homologous fragments and separating homologous fragments by point mutations can effectively reduce unfavorable recombination. These findings reveal a homologous recombination risk resulted from genome-homologous baculoviral elements and propose reliable strategies reducing recombination rate to facilitate viral stability and integrity in baculovirus expression vector system.

Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9.

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the N-glycosylation pathway including the presence of β(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for Arabidopsis thaliana and Nicotiana benthamiana. Such engineering of N-glycosylation would also be desirable for Nicotiana tabacum, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated N. tabacum cv. SR-1 β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four FucT and two XylT genes. These two enzymes are responsible for generating non-human N-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing β(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing Nicotiana tabacum as a biologics platform for Global Health.

A Novel Salt-Tolerant L-Glutaminase: Efficient Functional Expression, Computer-Aided Design, and Application

The low productivity in long fermentation duration and high-salt working conditions limit the application of L-glutaminase in soy sauce brewing. In this study, a novel L-glutaminase (LreuglsA) with eminent salt tolerance was mined and achieved more than 70% activity with 30% NaCl. To improve the robustness of the enzyme at different fermentation strategies, mutation LreuglsAH105K was built by a computer-aided design, and the recombinant protein expression level, an essential parameter in industrial applications, was increased 5.61-fold with the synthetic biology strategy by improving the mRNA stability. Finally, the LreuglsAH105K functional expression box was contributed to Bacillus subtilis 168 by auxotrophic complementation, and the production in a 5-L bioreactor was improved to 2516.78 ± 20.83 U mL−1, the highest production ever reported. When the immobilized cells were applied to high-salt dilute-state soy sauce brewing, the L-glutamate level was increased by 45.9%. This work provides insight into the salt-tolerant enzyme for improving the efficiency of industrial applications.

Evaluation of Phycocyanin Promoter Function in Bacteria by Investigating the Expression of HBsAg

Many items affect the yields of every recombinant protein production. Promoters are one of the key regulatory elements which control the level of recombinant protein expression in the host. Although in some studies (Hepatitis B Surface Antigen) HBsAg was cloned in E. coli, in many of them common promoters were used. In this study two co-vectors (PHK and PHGK) were designed and used. Both of them were shuttle types based on phycocyanin-specific promoter and each was a combination of two types of vectors: a plasmid and a transposon. ELISA results, in line with Western blot results, showed the ability of phycocyanin promoter as well as revealed the expression level of HBsAg in transgenes from the Top10 PHK vector is higher than those of Top10 PHGK. The expression of HBsAg under the phycocyanin promoter in the present study is 7.5 µg/lit. In the current study as a pilot step, our attitude and objective of bacteria expression are to evaluate the function of the phycocyanin promoter based on the HBsAg gene. Due to the nature of the shuttle vector, in continuation of the present study to reduce the non-responsive populations, high antigen expression, meet the need for renewal and, other benefits, expression of HBsAg in other than bacterial host is under investigation.

The Effect of microRNA on the Production of Recombinant Protein in CHO Cells and its Mechanism.

Recombinant protein production by mammalian cells is the initial step in the manufacture of many therapeutic proteins. Chinese hamster ovary (CHO) cells are the most common host system to produce recombinant therapeutic proteins (RTPs). However, it is still challenging to maintain high productivity ensuring the good quality of RTPs produced by CHO cells. MicroRNAs(miRNAs) are short regulatory non-coding RNAs that can regulate cellular behavior and complex phenotypes. It has been found that miRNAs can enhance the expression level of recombinant proteins in CHO cells by promoting proliferation, resisting apoptosis, and regulating metabolism. miRNAs also can affect the quality of RTPs. In this review, we will discuss the effect and mechanism of miRNA on the expression level and quality of recombinant proteins in CHO cells.

SOLUBILIZATION AND REFOLDING TECHNIQUES TO ATTAIN Candidatus liberibacter ASIATICUS OMP PROTEIN FROM INCLUSION BODIES

The OMP is a conserved and major outer membrane protein presented on the surface of Candidatus Liberibacter asiaticus (CLas). High expression level of recombinant outer membrane protein (OMP) in Escherichia coli led to form inclusion bodies (IBs) in cytoplasmic space which is an inactive form of protein and needs a solubilizing and refolding process. The aim of this study was to compare different methods for solubilization and refolding of recombinant OMP expressed in E. coli. For this aim, preliminary washing process was performed by Triton X-100 and sodium deoxycholate. To solubilize IBs, reducing agents; guanidine-hydrochloride, urea, β-mercaptoethanol and dithiothritol were applied. The results of washing process showed that applying of 0.5% TritonX-100 (v/v) is led to highest protein recovery. The results obtained from solubilization step revealed that applying guanidine-hydrochloride denaturing agent is led to obtain more solubilized protein. Furthermore, adding supplementary β-mercaptoethanol in denaturing buffer resulted to improve protein recovery during solubilization process. Presence of arginine in refolding buffer containing high urea concentration was led to significant decreases in aggregation. As a result, the best recovery of OMP was obtained at a concentration 0.5 M of Triton X-100, the high efficiency of solubility was found in pH 8 in the presence of guanidine-hydrochloride and the best efficiency of refolding was attained by final buffer containing 2M urea and 0.5 M L-Arginine. These results suggested a suitable procedure to produce appropriate recombinant OMP for preparation of poly and monoclonal antibodies.

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products.Key points• A protein expression vector using double-antibiotic resistance genes was designed• Double antibiotic selection causes fewer colonies with more positive for phenotype• Coupling the new vector with FACS improves microalgal screening efficiency > 60%

Construction of Yeast Display Libraries for Selection of Antigen-Binding Variants of Large Extracellular Loop of CD81, a Major Surface Marker Protein of Extracellular Vesicles.

Over the last two decades, yeast display methodology has served as a popular tool for discovery, humanization, stability improvement, and affinity maturation of antibodies and antibody fragments, but also for development of diverse non-antibody protein scaffolds towards the ability of antigen recognition. Yeast display is particularly well suited for multiparametric analysis of properties of derivatized proteins, allowing the evolution of most diverse protein structures into antigen binding entities with favorable expression, stability, and folding properties. Here we present the methodological basics of a novel yeast display-based approach for the functionalization of the large extracellular loop of CD81 into a de novo antigen binding unit. CD81 is intrinsically overrepresented on the surface of extracellular vesicles (EVs), naturally occurring nanoparticle units that act as cell-to-cell messengers by delivering their intracellular cargo from the source cell into a recipient cell. This amazing feature makes them of highest biotechnological interest, yet methods for their targeted delivery are still in their infancy. As a novel approach for introducing EV surface modifications enabling specific target cell recognition and internalization, we have prepared yeast display libraries of CD81 large extracellular loop mutants, which are selected towards specific antigen binding and resulting mutants conveniently clicked into the full-length EV surface protein. Resulting EVs display wild-type-like characteristics regarding the expression level and distribution of recombinant proteins and are hence promising therapeutic tools.

Developing a secretory AcGFP1-based IRES expression system for efficient production of mammalian recombinant proteins

To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening.

Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites.

Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann's ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during overexpression.

Michal Sharon

Angewandte ChemieVolume 133, Issue 36 p. 19673-19673 Introducing …Free Access Michal Sharon First published: 01 August 2021 https://doi.org/10.1002/ange.202108629AboutSectionsPDF ToolsRequest permissionAdd to favorites ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract “The biggest challenge facing scientists is navigating in the unknown … My favorite molecule is the proteasome complex …” Find out more about Michal Sharon in her Introducing … Profile. Michal Sharon The author presented on this page has published her first article as a submitting corresponding author in Angewandte Chemie: “Intracellular protein–drug interactions probed by direct mass spectrometry of cell lysates”: R. Rogawski, A. Rogel, I. Bloch, M. Gal, A. Horovitz, N. London, M. Sharon, Angew. Chem. Int. Ed. 2021, 60, doi.org/10.1002/anie.202104947; Angew. Chem. 2021, 133, doi.org/10.1002/ange.202104947. Position: Associate Professor, Department of Biomolecular Sciences, Weizmann Institute of Science Homepage: https://www.weizmann.ac.il/Biomolecular Sciences/MichalSharon/ ORCID: orcid.org/0000-0003-3933-0595 Education: 1996 B.Sc. in Chemistry, The Hebrew University, Jerusalem (Israel) 1996–2003 M.Sc./Ph.D. combined program (direct PhD) with Prof. Jacob Anglister, Department of Structural Biology, Weizmann Institute of Science, Rehovot (Israel) 2003–2006 Postdoc with Prof. Carol V. Robinson, Department of Chemistry, University of Cambridge, Cambridge (UK) Research: Structural mass spectrometry of proteins Hobbies: Hiking, reading, painting The biggest challenge facing scientists is navigating in the unknown. The most exciting thing about my research is that it never fails to surprise me going to unexpected directions. A turning point in my career was the decision to combine my chemical skills with biological questions. My long-term goal is continue and inspire young female scientists. I advise my students to believe in themselves. The most challenging part of my job is facing unfairness. I'm always in a good mood when I am inspired. To improve my work-life balance, I have wonderful friends. What I appreciate most about my friends is their endless support. The best advice I have ever been given is to be true to yourself. My biggest inspiration is my postdoc advisor Dame Professor Carol Robinson. The most important quality of a mentor is the ability to tailor support in a way that matches the student needs. My favorite thing about my lab group is that they are my second family. My favorite molecule is the proteasome complex. My favorite place on earth is the Israeli desert. Behind the Science References 1Recently, we developed a mass spectrometry (MS) methodology for in-depth, yet rapid, characterization of overproduced proteins without the need for protein purification. Although initially considered debatable, as prior purification of the protein complex of interest was considered a prerequisite for acquiring mass spectra. We have shown that it is possible to rely on the high expression levels of recombinant proteins and circumvented this tedious and laborious need. We wanted to adapt this direct-MS method to proteins generated intracellularly in human cells but this was extremely challenging. After multiple failures, we decided to adapt a system used during in-cell NMR studies, which turned out to solve the technical problems and pave the way for this study. Google Scholar Volume133, Issue36September 1, 2021Pages 19673-19673 This is the German version of Angewandte Chemie. Note for articles published since 1962: Do not cite this version alone. Take me to the International Edition version with citable page numbers, DOI, and citation export. We apologize for the inconvenience. ReferencesRelatedInformation