Step-by-Step Preparation of Immobilized Recombinant Staphylococcus aureus Protein A (SpA): A Versatile Tool for Efficient Antibody Purification.

Cloning, expression, and purification of HpaA-CagA fusion recombinant protein of Helicobacter pylori in E. coli BL 21 strain

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

Magnetic nanoparticles (MNPs) with an amine functionalized surface (MH) were passivated with carbonaceous materials (MH@C) by carbonization of glucose under hydrothermal reaction conditions. The carboxylate groups in carbonaceous shell could be enriched to 0.285 mmol/g when acrylic acid was added as a functional monomer in the carbonization reaction (MH@C-Ac). The carbonaceous shell not only protected the magnetic core from acidic erosion but also showed a high adsorption capacity toward Ni(2+) ion. The Ni(2+) ion complexed on MH@C and MH@C-Ac could specifically isolate 6×His tagged recombinant proteins from crude bacterial extracts via metal affinity interaction. The superparamagnetic property facilitates the easy retrieval of the carbonaceous material passivated MNPs from the viscous proteins solutions. Recombinant green fluorescence protein (GFP) and hyaluronic acid (HA) lyase of 9.4 mg and 2.3 mg could be isolated by 1 g of MH@C-Ac-Ni, respectively.

Wild type p53 exists in a constant state of equilibrium between wild type and mutant conformation and undergoes conformational changes at elevated temperature. We have demonstrated that the co-chaperone CHIP (carboxyl terminus of Hsp70-interacting protein), which suppressed aggregation of several misfolded substrates and induced the proteasomal degradation of both wild type and mutant p53, physically interacts with the amino terminus of WT53 and prevented it from irreversible thermal inactivation. CHIP preferentially binds to the p53 mutant phenotype and restored the DNA binding activity of heat-denatured p53 in an ATP-independent manner. In cells under elevated temperatures that contained a higher level of p53 mutant phenotype, CHIP restored the native-like conformation of p53 in the presence of geldanamycin, whereas CHIP-small interfering RNA considerably increased the mutant form. Further, under elevated temperatures, the levels of CHIP and p53 were higher in nucleus, and chromatin immunoprecipitation shows the presence of p53 and CHIP together upon the DNA binding site in the p21 and p53 promoters. We propose that CHIP might be a direct chaperone of wild type p53 that helps p53 in maintaining wild type conformation under physiological condition as well as help resurrect p53 mutant phenotype into a folded native state under stress condition.

Interest in magnetic nanoparticles for capturing bacteria arises from a variety of attributes, including the similar size of nanoparticles, magnetic behavior, and attached biomolecules such as proteins and nucleotide probes. Here we report the application of amine-functionalized magnetic nanoparticles (AF-MNPs) for rapid and efficient capture and removal of bacterial pathogens. The AF-MNPs are used without the need for any further modifications with affinity biomolecules. The positive charges on the surface of AF-MNPs can promote strong electrostatic interaction with negatively charged sites on the surface of bacterial pathogens to exhibit efficient adsorptive ability. The hydrophobic interaction between the pendant propyl group of the amine functionality and the bacteria also plays a supplementary role. The amine groups on the surface of the magnetic nanoparticle are robust and inexpensive ligands to ensure a high binding affinity to at least eight different species of Gram-positive and Gram-negative bacteria. The amount of AF-MNPs, pH of phosphate buffer solution, and ionic strength are crucial in mediating fast and effective interactions between AF-MNPs and bacteria. The AF-MNPs allow rapid removal of bacteria from water samples, food matrixes, and a urine sample with efficiency from 88.5% to 99.1%. Though amino group offers less specificity/selectivity than biomolecules such as antibodies, AF-MNPs are attractive for capturing a wide range of bacteria.

On-chip enzymatic microreactor using trypsin-immobilized superparamagnetic nanoparticles for highly efficient proteolysis

BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study.ResultsWe firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers.ConclusionsIn this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0268-7) contains supplementary material, which is available to authorized users.

Facile preparation of magnetic carbonaceous nanoparticles for Pb 2+ ions removal

Human papillomavirus (HPV) is the leading cause of cervical cancer worldwide. The pathogenesis of HPV is mainly dependent on its E7 and E6 proteins. Up to now, different adjuvants have been used to enhance the efficacy of the immune response against these two proteins. In this study, Flagellin (FLA) was used as adjuvant to test adjuvant activity and also see whether its orientation of attachment can affect the immune response pattern. The E7d-FLA and FLA-E7d in pET28a vector were constructed and then the recombinant proteins were expressed in E. coli BL21 (DE3) bacteria under IPTG induction. The expression of recombinant E7d-FLA and FLA-E7d proteins is confirmed by SDS-PAGE and western blot. Then, recombinant fusion proteins were purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. The recombinant proteins were checked for endotoxin contamination and then quantified by Bradford. Eight-to-ten-week-old male Balb/C mice were immunized subcutaneously with 10 µg recombinant E7d-FLA, FLA-E7d and HPV16E7d vaccine on days 0, 14 and 28. In addition, PBS and FLA groups were considered as control group. Then, spleen cells were harvested to assess lymphocyte proliferation and IFN-γ, IL-4 and IL-17 cytokines. In addition, mice sera were used for specific total IgG and IgG1, IgG2a, IgG2b and IgM antibodies assessment by ELISA. The results show that E7d-FLA is more potent in the induction of lymphocyte proliferation, CTL response and specific total IgG, IgG2a and IgG2b response, while the FLA-E7d vaccine was associated with more IFN-γ, and IL-17 cytokine response. The results of this study proved the ability of FLA as an adjuvant in fusion with E7d in the induction of cellular and humoral immune responses. In addition, it also emphasizes that antigen-adjuvant orientation can affect the immune response strength and polarization against HPV E7d vaccine candidate. HIGHLIGHTS: Flagellin is attached to HPV-16 E7d at the C- or N-terminus to create E7d-FLA and FLA-E7d candidate vaccines. The E7d-FLA vaccine showed a significant increase in lymphocyte proliferation, CTL response and IgG response versus FLA-E7d vaccine. The FLA-E7d vaccine is associated with a significant increase in IFN-γ and IL-17 cytokines response versus E7d-FLA vaccine. It seems that that antigen-adjuvant orientation is an important parameter in the strength and polarization of immune response in HPV E7d vaccine candidate.

The removal of highly stable dispersed oil produced during oil recovery processes is very challenging, especially in offshore operations where the limited space does not allow use of equipment with long residence time for the required separation. Using magnetic nanoparticles (MNPs) to remove the dispersed oil from produced water is a promising way to overcome the difficulties that the current treatment technologies face, since the MNPs-attached oil droplets can be quickly and efficiently separated with application of an external magnetic field. The MNPs can be also regenerated and reused, minimizing the generation of hazardous waste. We investigated not only the optimal operating conditions, such as MNP concentration and salinity, but also the mechanisms of MNPs-oil attachment and magnetic separation.We synthesized MNPs in the laboratory with a prescribed surface coating. The MNPs were superparamagnetic with an average individual particle size of ~10 nm. Crude oil content in separated water was reduced by as much as 99.9% using MNP concentrations as low as 0.04 wt% in 5 minutes after MNPs and oil were reacted.The electrostatic attraction between negatively charged oil-in-water emulsions and positively charged MNPs controls the attachment of MNPs to the droplet surface; and the subsequent aggregation of the electrically neutral MNPs-attached oil droplets plays a critical role for accelerated and efficient magnetic separation. The particle aggregation occurred fast, generally within one minute. Thus, the total magnetic separation time was dramatically reduced to as short as 1 second, contrary to that of free, individual MNPs where it took about 36~72 hours, depending on the MNP concentrations.Model calculations of magnetic separation velocity, accounting for the MNP magnetization and viscous drag, show that the velocity of free Amine functionalized MNPs (A-MNPs) increases about 1~3 orders of magnitude as the particles get closer to the magnet depending on the particle size. The smaller the particles, the greater the effect of the magnetic field on the velocity. A typical operating condition would be when the size of the MNPs-oil droplet aggregates is grown to be greater than 360 nm. Then, the total magnetic separation time will be approximately 5 minutes.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Establishment of purification method for prokaryotic expression of Serpin gene for Dermatophagoides farinae

“What is the point of learning this?” You may have heard this question from students before and tried to answer it by providing some real-world applications of the concept at hand. Providing relevance to students' current academic lives and interests, their future professional lives, and real-world situations can help establish the value of course content and improve student learning 1. Infusing courses with social and even historical context can also improve student learning and retention 2. Activities designed to provide real-world case studies and to showcase the importance of science in students' lives have been developed for physiology courses (“physiological curiosity of the week” 3) and introductory biology (“disease of the week” 4), among many others, and have been shown to improve student engagement and interest in the subject matter. These activities were designed to improve students' perceptions of medical case studies, enhance their self-directed learning 3, and develop their writing abilities 4. To provide a realistic view of the biotechnology industry for students, a novel course focusing on recombinant proteins and their importance in medicine, pharmaceuticals, industry, scientific research, and agriculture was developed. “Designer Proteins and Society,” an upper-division elective, was taught in the Fall 2012 semester to 16 junior, senior, and first-year master's students in the Department of Biology at North Carolina A&T State University in Greensboro, NC. The major goal of the course was to challenge students to develop proposals that contained the steps required to design and produce recombinant proteins starting from RNA and included the methods of molecular cloning, recombinant protein expression and purification, and experiments to assess the function of the recombinant protein. Students also completed a five-week laboratory project based on the Bio-Rad Biotechnology Explorer Protein Expression and Purification Series (Bio-Rad, Hercules, CA) 5 that provided them hands-on experience with recombinant protein techniques through the expression, purification, and functional assessment of a recombinant form of human dihydrofolate reductase, an enzyme involved in the synthesis of nucleic acid precursors. The course ended with a field trip to a local biomanufacturing company, where students observed the production of therapeutic recombinant proteins at industrial scale. A key learning objective of the course was for students to explain how recombinant proteins are used in medical, pharmaceutical, industrial, and scientific research applications and why they are so important to society. To expose students to the myriad of commercially available recombinant proteins that are used as therapeutic drugs (e.g. insulin for diabetes treatment), food processing enzymes (e.g. chymosin used in cheese production), and molecular biology workhorses (e.g. Taq polymerase for PCR) and how essential they are to our daily lives, the “Recombinant Protein of the Day” assignment was developed and implemented. This assignment provided students with opportunities to research the scientific and commercial literature and practice their oral presentation skills, all while taking ownership of a particular recombinant protein that they found specific interest in. In the “Recombinant Protein of the Day” assignment, students were instructed to search the web to find any commercially available recombinant protein and were given a worksheet that included a list of required information they were to research about their protein. If they had trouble finding recombinant proteins for the assignment, they were instructed to ask for help. The required information included the name and species isoform of the protein, a brief description of the biological function of the protein, number of amino acids, molecular weight, isoelectric point, a picture of the crystal structure (if available), how society uses this protein (e.g. as a therapeutic drug), the name of the manufacturer, when the protein was first produced, and any information about the production process (especially that concerning the expression system and purification methods). Once students acquired this information about their recombinant protein, they developed a single PowerPoint slide to use for a short (2–4 min) presentation. The assignment worksheet that was handed out to students is included in Supporting Information. Students were also given a grading rubric (also in Supporting Information) that outlined the point distribution for this assignment. Each student was required to research and present on one recombinant protein during the semester. This assignment was worth 5% of their total course grade. At the beginning of each day of class for the first two-and-a-half months of the semester, the first 5–10 min were devoted to the “Recombinant Protein of the Day” presentations. The instructor gave the first three presentations in order to model examples of appropriate content. Students then gave their presentations daily, with no more than two students presenting per day. The nonpresenting students in the class were encouraged to ask questions of their classmates after their presentations were over. Students chose a wide variety of recombinant proteins to investigate, but mostly focused on recombinant proteins used as pharmaceutical drugs (Table 1). Several students had personal connections to their choice of protein, including having a relative or friend diagnosed with a disease relating to the protein, or being interested in studying the protein or disease during graduate or professional school. The “Recombinant Protein of the Day” presentations yielded a unique (and often unpredictable) beginning to each day of class, which also opened up discussions of aspects of recombinant proteins that were not covered in detail elsewhere in the course (such as fusion proteins, the difference between biological and pharmaceutical drugs, and specific therapeutic applications of recombinant proteins). Students performed well on the “Recombinant Protein of the Day” activity, with scores ranging from 80 to 100%. Students commonly missed points for failing to include all the required components or for lacking details about specific aspects of the activity (e.g. missing details about the biological function of the protein and how the recombinant protein is expressed or purified). Students were not held accountable (through summative assessments) for the content material in these presentations, as the goal of this activity was to expose students to the multitude of ways that recombinant proteins positively impact our society and not to assess student learning of the details of each recombinant protein that was presented. To measure the impact of the “Recombinant Protein of the Day” activity on student outcomes, students completed a pretest on the first day of class and a posttest on the last day of class. The pretest and posttest assessed students' content knowledge as well as their self-perceived abilities to perform specific tasks related to the course. This test was modeled on the instrument described by Bellin et al. 6 and was reviewed and found to be exempt by the North Carolina A&T State University Institutional Review Board. One question asked students to self-assess their confidence in their abilities to “explain how recombinant proteins are used by society” using a Likert-type scale of 0–5, with a value of zero being “not confident at all” and a value of five being “extremely confident.” As shown in Fig. 1, on the pretest students responded with low-confidence (scores of 0, 1, 2, or 3) in their abilities to explain how recombinant proteins are used by society. However, on the posttest, students responded with high-confidence (scores of 3, 4, or 5) in their ability to perform this task. While it is not possible to attribute this shift to higher student confidence to explain how recombinant proteins are used by society solely to the “Recombinant Protein of the Day” activity, it is likely that this activity played a significant role in this shift. Other components of the course addressed how recombinant proteins are used by society, but none were as explicit or student-centered as the “Recombinant Protein of the Day” activity. Student self-assessment of their abilities to “explain how recombinant proteins are used by society” compared from a pretest to a posttest. A value of zero indicates “not confident at all” and a value of five indicates “extremely confident.” n = 10 (pretest), n = 13 (posttest). At the end of the semester, students completed a course evaluation that asked them to rate different components of the course in terms of how much each component contributed to learning the course material. Students responded on a Likert-type scale of 1–5, with a value of one being “I learned very little or nothing at all” and a value of five being “I learned a lot.” Ten of the 13 students that completed the evaluation assigned the “Recombinant Protein of the Day” assignment a score of four or five (Fig. 2), indicating that students thought that the assignment greatly contributed to their learning during the course. In additional, several written student comments indicated that they appreciated and enjoyed the real-world aspects of the course. For example, when asked about what aspects of the course were most valuable, one student remarked “learning how the lessons we were taught are being used in everyday life.” Student ratings of the “Recombinant Protein of the Day” assignment in terms of how much this assignment contributed to their learning the course material. A value of one indicates “I learned very little or nothing at all” and a value of five indicates “I learned a lot.” n = 13. In summary, daily presentation assignments such as “Recombinant Protein of the Day” may be useful in stimulating student interest, providing opportunities for students to develop oral presentation skills and in bringing real-world connections to classrooms in a student-centered manner. Assignments such as this could be easily modified to suit any course content (chemical reaction of the day, gene of the week, etc.) to motivate students to pursue personally relevant topics that may pique their interest and engage them in the learning process. The author would like to thank Brian Rybarczyk and Jennifer Tenlen for critical reading of this manuscript. This work was supported by the Seeding Postdoctoral Innovators in Research and Education (SPIRE) program funded by the National Institutes of Health Training, Workforce Development, and Diversity division of the National Institute of General Medical Sciences (K12GM000678). Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron

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.