Optimized Expression System Research Articles

Biosensing strategies using recombinant luminescent proteins and their use for food and environmental analysis.

Progress in synthetic biology and nanotechnology plays at present a major role in the fabrication of sophisticated and miniaturized analytical devices that provide the means to tackle the need for new tools and methods for environmental and food safety. Significant research efforts have led to biosensing experiments experiencing a remarkable growth with the development and application of recombinant luminescent proteins (RLPs) being at the core of this boost. Integrating RLPs into biosensors has resulted in highly versatile detection platforms. These platforms include luminescent enzyme-linked immunosorbent assays (ELISAs), bioluminescence resonance energy transfer (BRET)-based sensors, and genetically encoded luminescent biosensors. Increased signal-to-noise ratios, rapid response times, and the ability to monitor dynamic biological processes in live cells are advantages inherent to the approaches mentioned above. Furthermore, novel fusion proteins and optimized expression systems to improve their stability, brightness, and spectral properties have enhanced the performance and pertinence of luminescent biosensors in diverse fields. This review highlights recent progress in RLP-based biosensing, showcasing their implementation for monitoring different contaminants commonly found in food and environmental samples. Future perspectives and potential challenges in these two areas of interest are also addressed, providing a comprehensive overview of the current state and a forecast of the biosensing strategies using recombinant luminescent proteins to come.

Purification and characterization of soluble recombinant Crimean-Congo hemorrhagic fever virus glycoprotein Gc expressed in mammalian 293F cells

BackgroundCrimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies.MethodsWe designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen.ResultsThe optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation.ConclusionsThe purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen’s suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.

A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves

BackgroundOptimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens‑mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported.ResultsWe developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB.ConclusionThis study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach.

Abstract 1660 Designing an optimal expression system of peptides in E. coli

Abstract 1660 Designing an optimal expression system of peptides in E. coli

Development of an Efficient Recombinant Protein Expression System in Clostridium saccharoperbutylacetonicum Based on the Bacteriophage T7 System.

Recombinant proteins have broad applications. However, there is a lack of a recombinant protein expression system specifically for large-scale production in anaerobic hosts. Here, we developed a powerful and stringently inducible protein expression system based on the bacteriophage T7 system in the strictly anaerobic solvent-producing Clostridium saccharoperbutylacetonicum. With the integration of a codon optimized T7 RNA polymerase into the chromosome, a single plasmid carrying a T7 promoter could efficiently drive high-level expression of the target gene in an orthogonal manner, which was tightly regulated by a lactose-inducible system. Furthermore, by deleting beta-galactosidase genes involved in lactose metabolism, the transcriptional strength was further improved. In the ultimately optimized strain TM-07, the transcriptional strength of the T7 promoter showed 9.5-fold increase compared to the endogenous strong promoter Pthl. The heterologous NADP+-dependent 3-hydroxybutyryl-CoA dehydrogenase (Hbd1) from C. kluyveri was expressed in TM-07, and the yield of the recombinant protein reached 30.4-42.4% of the total cellular protein, surpassing the strong protein expression systems in other Gram-positive bacteria. The relative activity of Hbd1 in the crude enzyme was 198.0 U/mg, which was 8.3-fold higher than the natural activity in C. kluyveri. The relative activity of the purified enzyme reached 467.4 U/mg. To the best of our knowledge, this study represents the first application of the T7 expression system in Clostridium species, and this optimized expression system holds great potential for large-scale endotoxin-free recombinant protein production under strictly anaerobic conditions. This development paves the way for significant advancements in biotechnology and opens up new avenues for industrial applications.

Evaluation of different expression systems for the heterologous production of sprivivirus glycoprotein

Glycoprotein mediated the entry of rhabdoviruses into the host cell and was the target of neutralizing antibodies. It was a prime tool for the study of the evolution and pathogenesis of viruses. Here, four different expression systems including cell-free synthesis system, Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda were used to explore the recombinant expression of spring viraemia of carp virus glycoprotein. The results indicated that the recombinant glycoprotein could be detected in E. coli cell-free system after in vitro incubation for 16 h at 25 °C. E. coli was found to be the optimal expression system for the production of the largest amount of recombinant glycoprotein. Although robust yields were considerable in E. coli, the formation of insoluble aggregates and the removal of solubilizing labels were challenging for the expression and purification of glycoprotein. For this reason, the higher eukaryotic expression systems, yeast GS115 and insect Sf9 cells were used for the production of glycoprotein. The purity of the recombinant protein obtained from yeast culture was over 90% after purification by Ni-NTA. In insect cells, the target protein was expressed mainly in the intracellular environment, but secretory glycoprotein was also detected by western blotting. These findings provided a basis for further investigations into the structure and function of SVCV glycoprotein.

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells.

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

Functional characterization of glutamate carboxypeptidase II: a multifunctional Zn‐metalloprotease

Glutamate Carboxypeptidase II (GCPII) is a dimeric, transmembrane, zinc metallopeptidase produced in many organisms including humans. Human GCPII (hGCPII) is expressed in the brain, prostate, intestine, and kidney, and is therefor predicted to exhibit varying tissue‐specific hydrolase activities. The only documented GCPII hydrolase activities are the breakdown of N‐acetyl‐aspartyl‐glutamate (NAAG) – GCPII is involved in the development of neurotoxicity due to excess glutamate – and folyl‐poly‐γ‐glutamate – GCPII is involved in folate uptake across intestinal mucosal membrane. However, functions in other tissues have not been reported. A comprehensive understanding of potentially diverse tissue‐specific proteolytic and other biochemical functions of GCPII requires detailed molecular level in vivo and in vitro studies using the full‐length GCPII and suitable model organism respectively.Homology modeling and protein structure analyses identified a hGCPII ortholog in the roundworm Caenorhabditis elegans, (hereafter referred as cGCPII), thus C. elegans has been chosen for the in vivo studies. The hGCPII and the cGCPII share all essential protein structural features. The cGCPII is encoded as three paralogs (gcp‐2.1, gcp‐2.2, gcp‐2.3) compared to five paralogs in humans. Whole animal phenotypic studies carried out using the wild‐type (N2) and the mutant RB1055, which lacks the gcp‐2.1, showed a larger brood size for RB1005 than N2 and longevity assays showed similar lifespans for both strains. The N2 and RB1055 strains raised in the presence of the folate hydrolase inhibitor sulfasalazine (SSZ) and GCPII specific inhibitors 2‐(Phosphonomethyl)pentane‐1,5‐dioicacid (PMPA) or 2‐(3‐Mercaptopropyl)pentanedioic acid (2‐MPPA) and the showed significantly reduced brood size for RB1055 compared to N2. The functional roles of individual cGCPII paralogs were investigated by RNAi mediated knockout experiments. Collective results indicate that the all three cGCPII paralogs may be involved in functional complementation in C. elegans. For the in vitro studies, the full‐length hGCPII was cloned and overexpression was carried out in Pichia pastoris. Several hGCPII expressing transformants have been identified and expression optimization is underway. Parallel cloning in insect cells in process to compare the functionally important glycosylation levels in Pichia versus the insect cells in order to choose an optimal expression system. Together, the in vivo and in vitro findings will aid in further understanding of the physiological functions of GCPII.Support or Funding InformationEastern Illinois UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Engineering of Corynebacterium glutamicum for Consolidated Conversion of Hemicellulosic Biomass into Xylonic Acid.

Xylonic acid is a promising platform chemical with various applications in the fields of food, pharmaceuticals, and agriculture. However, in the current process, xylonic acid is mainly produced by the conversion of xylose, whose preparation requires substantial cost and time. Here, Corynebacterium glutamicum is engineered for the consolidated bioconversion of hemicellulosic biomass (xylan) into xylonic acid in a single cultivation. First, for the efficient conversion of xylose to xylonic acid, xylose dehydrogenase (Xdh) and xylonolactonase (XylC) from Caulobacter crescentus are evaluated together with a previously optimized xylose transporter module (XylE of Escherichia coli), and cells expressing xdh and xylE genes with an optimized expression system can produce xylonic acid from xylose with 100% conversion yield. Next, to directly process xylan as a substrate, an engineered xylan-degrading module is introduced, in which two xylan-degrading enzymes (endoxylanase and xylosidase) are secreted into the culture medium. The engineered C. glutamicum successfully produce 6.23 g L-1 of xylonic acid from 20 g L-1 of xylan. This is the first report on xylonic acid production in C. glutamicum and this robust system will contribute to development of an industrially relevant platform for production of xylonic acid from raw biomass.

Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights.

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. FTP is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically active recombinant FTP in Escherichia coli BL21, optimizing conditions with the best set yielding >40 mg/l culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/ml of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP–mannose and FTP–glucose crystals were obtained, and they diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å respectively. The best solution showed four monomers per asymmetric unit. Molecular dynamics (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was also capable of stimulating cell migration at 50 µg/ml. In conclusion, our optimized expression system allowed high amounts of correctly folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example in wound healing and tissue regeneration.

Not Limited to E. coli: Versatile Expression Vectors for Mammalian Protein Expression.

Recombinant protein expression is not limited to E. coli or other prokaryotic systems. It is inevitable to use eukaryotic systems in order to express challenging mammalian proteins. Eukaryotic systems are able to perform complex posttranslational modifications like protein processing, phosphorylation, glycosylation, which are essential for stability and functionality of many proteins. Different eukaryotic protein expression systems employing yeast, insect, or mammalian cell lines are established with each having its own advantages and disadvantages. Often it is quite difficult to decide which will be the most optimal expression system as this depends highly on the protein itself. Expression in stable cell lines requires substantial screening of expressible constructs prior to developing a stable expression cell line. To achieve fast screening by transient expression in multiple hosts, versatile vectors can be applied. In this chapter, we present an overview of the most common multi-host vectors, which allow for fast expression analysis without tedious (re)cloning of the gene of interest in several different protein production systems. The protocols in this chapter describe the latest methods for fast transient expression in insect and mammalian cell lines.

Virus-Enabled Optimization and Delivery of the Genetic Machinery for Efficient Unnatural Amino Acid Mutagenesis in Mammalian Cells and Tissues.

Unnatural amino acid (UAA) mutagenesis of recombinant proteins in live mammalian cells requires coexpression of the mutant target, as well as an engineered tRNA/aminoacyl-tRNA synthetase pair. The ability to readily determine the optimal relative expression levels of these three genetic components for efficient expression of the UAA-modified target is highly desirable, but remains challenging to accomplish. Here we report a facile strategy to achieve this by taking advantage of the efficient gene-delivery by a baculovirus vector, which enables systematic variation of the expression level of each genetic component in a population-wide manner. Insights gained from this study led to the design of an optimal expression system, which can be delivered into mammalian cells by a single baculovirus vector to provide significantly improved UAA incorporation efficiency at a low virus load. Furthermore, this optimized baculovirus vector was shown to enable efficient UAA mutagenesis of proteins expressed in mouse brain tissue.

Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli

Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L.

Optimized expression and purification of biophysical quantities of Lac repressor and Lac repressor regulatory domain

The recombinant production of Lac repressor (LacI) in Escherichia coli is complicated by its ubiquitous use as a regulatory element in commercially-available expression vectors and host strains. While LacI-regulated expression systems are often used to produce recombinant LacI, the product can be heterogeneous and unsuitable for some studies. Alternative approaches include using unregulated vectors which typically suffer from low yield or vectors with promoters induced by metabolically active sugars which can dilute isotope labels necessary for certain biophysical studies. Here, an optimized expression system and isolation protocol for producing various constructs of LacI is introduced which eliminates these complications. The expression vector is an adaptation of the pASK backbone wherein expression of the lacI gene is regulated by an anhydrotetracyline inducible tetA promoter and the host strain lacks the lacI gene. Typical yields in highly deuterated minimal medium are nearly 2-fold greater than those previously reported. Notably, the new expression system is also able to produce the isolated regulatory domain of LacI without co-expression of the full-length protein and without any defects in cell viability, eliminating the inconvenient requirement for strict monitoring of cell densities during pre-culturing. Typical yields in highly deuterated minimal medium are significantly greater than those previously reported. Characterization by solution NMR shows that LacI constructs produced using this expression system are highly homogenous and functionally active.

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms.

Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme's role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.

Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

SAT0256 Analytical and Functional Assessments When Developing Biosimilar Candidates

BackgroundBiosimilar monoclonal antibodies (mAb) are expressed in unique host cell systems and without knowledge of specific process conditions, which are proprietary to the innovator. A biosimilar developer may choose to...

Preparation of recombinant human canstatin using transgenic <italic>Dunaliella salina</italic>

Canstatin, which possesses a significant inhibition effect on the migration of endothelial cells and a strong anticancer effect [1,2], has been applied in the treatment of many cancers including human oral, breast, prostate, pancreatic, and colorectal [3–7]. However, because the expression of bioactive recombinant canstatin is very low using the current expression systems, e.g. prokaryotic Escherichia coli expression system [6], its application has still been limited to clinical trials. Several eukaryotic cell expression systems have been exploited for canstatin production, such as Bombyx mori cells [8] and Drosophila melanogaster S2 cells [9], but they also have a lot of disadvantages, for example, high culture cost, poor yield, and difficulty in purification. Therefore, it is necessary and urgent to develop an optimal expression system for the large-scale production of the recombinant canstatin. Transgenic Dunaliella salina (UTEX-1644) system as a novel potential bioreactor [10,11] is an optimal alternative for the production of the recombinant canstatin due to the following advantages: (i) this expression system has the potential for large-scale production of exogenous proteins; (ii) D. salina has been extensively used in industrial and pharmaceutical areas owing to the capability of accumulating valuable fine components such as carotenoids, vitamins, minerals, and proteins; (iii) D. salina cells themselves are natural protoplasts, and can be easily transformed and cultured; (iv) eukaryotic D. salina cells have the post-transcriptional and post-translational modifications for the production of bioactive proteins. In the present study, therefore, we tried to develop a novel eukaryotic expression system for the recombinant canstatin by using transgenic D. salina, and this system will provide a new, safe, and environmental protection platform for the large-scale production of human recombinant canstatin. The main materials used in this study were as follows: plasmids pBI221-bar and pUV-GUS were obtained as a gift from Prof. Yongru Sun (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China). D. salina strain UTEX-1644 was purchased from the Algae Culture Collection at the University of Texas (Austin, USA) and cultured in the modified PKS medium under a light intensity of 50 mmol photon m s with a 12/12-h light/day [12]. To amplify the canstatin gene, the total RNA of human placental tissues was isolated. The Primer 1 (50-ACTCCC GGGGTCAGCATCGGCTACCTCCT-30) and Primer 2 (50-CCGAGCTCTCAATGGTGATGGTGATGGTG CA GGTTCTTCATGCACAC-30) were designed in which the Sma_ and Sac_ sites (underlined) were introduced respectively (the bold sequence represents the sequence of His6). Using the above RNA and primers, the human canstatin gene was amplified by reverse transcription–polymerase chain reaction (RT–PCR) and the results showed that a specific fragment of 700 bp was successfully amplified. The results of sequencing indicated that the amplified DNA fragment was completely identical to the nucleotide sequence reported in GenBank. Then, the canstatin gene was inserted into pMD18-T (TaKaRa, Dalian, China) to yield a new plasmid pMD18-T-Can for plasmid propagation and DNA sequencing. Subsequently, the canstatin fragment cut from the pMD18-T-Can by digestion of endonucleases was inserted into pUV-GUS to generate a novel vector pUV-Can. After being confirmed by digestion of double enzymes, the pUV-Can was recovered and further connected with the bar box to generate a eukaryotic expression vector pUV-Can-Bar. pUV-Can-Bar vectors were transformed into the D. salina cells using the glass beads method [12], and then the transformed D. salina cells were incubated for 24 h under dim light conditions. After the transformed D. salina cells were incubated for 2 weeks on a 1% agar plate containing 3 mg/l of phosphinothricin (PPT), the individual positive colonies were observed, among which four colonies were picked out and inoculated into the liquid medium with 3 mg/l of PPT for further selection. The resistant test of the transformants demonstrated that all the transformants survived from the following Acta Biochim Biophys Sin 2014, 46: 428–430 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu009. Advance Access Publication 23 March 2014

The production of recombinant 15N, 13C-labelled somatostatin 14 for NMR spectroscopy

Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer polypeptide product lowered the yield of the target peptide and complicated its purification. The side product contained the N-terminal 6His-tag together with the thioredoxin fusion partner and the specific enzymatic cleavage site-containing linker followed by an unknown peptide starting with the first 7N-terminal amino acid residues of SS14, as revealed by the Edman degradation. The combination of DNA sequence analysis, the Edman degradation, and high-resolution mass spectrometry allowed to identify the amino acid sequence of the unknown peptide. The appearance of the side product was attributed to inefficient termination of mRNA translation. The stop codon and its downstream sequence optimization allowed eliminating the side product synthesis. The optimized expression system, purification protocol, and post-translational modification procedure yielded 1.5mg of SS14 per liter of minimal medium. Nearly 99% incorporation of 13C and 15N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.