Exploration of an online and offline mixed teaching mode for "Protein Engineering" based on BOPPPS+flipped classroom / 生物工程学报

Protein Engineering is a core compulsory course of biotechnology major, which is the first-class undergraduate major being constructed in Shanxi Province. In view of the problems of single teaching mode of Protein Engineering, such as insufficient students' participation, short teaching time, and expensive experiment cost, the course team carried out the reform and practice of teaching mode for this course, and put forward a new teaching strategy. Under the guidance of the "Golden Course" standard for advancement, innovation and challenge, the course team developed the materials for massive open online courses (MOOC), and carried out the online and offline mixed teaching of Protein Engineering based on BOPPPS+flipped classroom by using the Chao-Xing Fan-Ya network teaching platform. Through this, a comprehensive, systematic and dynamic new teaching system of Protein Engineering was developed. Using the teaching mode based on BOPPPS+flipped classroom, the offline classroom teaching was combined with students' online self-study and homework completion, chapter test and discussion, and this mixed teaching mode was fully integrated into the flipped classroom. After three rounds of teaching practice, the course team had developed a complete, reproducible, scientific and reasonable online and offline mixed teaching mode, which included course materials preparation, exploring experiment guidance, classroom discussion design and course performance evaluation. The online and offline mixed teaching mode of Protein Engineering based on BOPPPS+flipped classroom was helpful for students to improve their autonomous learning ability, to be deeply engaged in the whole teaching process, and to develop a comprehensive and profound understanding of Protein Engineering. This teaching mode improved the teaching quality of Protein Engineering, and facilitated students to learn other follow-up professional courses. Moreover, it provides a reference for the course teaching reform.

Recent advances in neonatal Fc receptor targeted drug delivery systems / 药学学报

The neonatal Fc receptor (FcRn) was first found to be a membrane protein that maternal antibodies transmitted to fetuses and newborns, and also expressed in multiple organs and tissues for whole life in adults. It plays a significant role to central regulate the lifespan of immunoglobulin G and serum albumin, as well as its involvement in innate and adaptive immune responses. In modern biopharmaceuticals, FcRn is a great potential drug delivery target and a highlighted subject for current research. This paper briefly describes the basic biological properties and action mechanism of FcRn, as well as the commonly used drug carrier design strategies of FcRn, especially the functional applications of prolonging half-life, targeted drug delivery, transmembrane and antigen presentation and so on. We propose that these distribution in different tissues and the diverse biological activities may have significant implications of targeting FcRn for novel drug delivery systems and immunotherapy.

Effect of key enzymes ubiquitination sites on the biosynthesis of naringenin / 生物工程学报

Flavonoids have a variety of biological activities and have important applications in food, medicine, cosmetics, and many other fields. Naringenin is a platform chemical for the biosynthesis of many important flavonoids. Ubiquitination plays a pivotal role in the post-translational modification of proteins and participates in the regulation of cellular activities. Ubiquitinated proteins can be degraded by the ubiquitin-protease system, which is important for maintaining the physiological activities of cells, and may also exert a significant impact on the expression of exogenous proteins. In this study, a real-time in-situ detection system for ubiquitination modification has been established in Saccharomyces cerevisiae by using a fluorescence bimolecular complementation approach. The ubiquitination level of protein was characterized by fluorescence intensity. By using the approach, the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway have been obtained. The lysine residues of the relevant ubiquitination sites were mutated to arginine to reduce the ubiquitination level. The mutants of tyrosine ammonia-lyase (FjTAL) and chalcone synthase (SjCHS, SmCHS) showed decreased fluorescence, suggested that a decreased ubiquitination level. After fermentation verification, the S. cerevisiae expressing tyrosine ammonia-lyase FjTAL mutant FjTAL-K487R accumulated 74.2 mg/L p-coumaric acid at 72 h, which was 32.3% higher than that of the original FjTAL. The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin. The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.

Usos de la levadura Pichia pastoris en la producción de proteínas recombinantes / Uses of Pichia pastoris yeast in the production of recombinant proteins

La levadura metilotrófica Pichia pastoris (clasificada actualmente como Komagataella phaffii) es una de las más importantes para la producción de proteínas heterólogas. En el trabajo se presenta un análisis de las principales características que se ponen de manifiesto en la expresión de proteínas recombinantes expresadas en este microorganismo. Se describen las cepas disponibles para la transformación y producción de proteínas recombinantes expresadas en Pichia pastoris, los principales vectores comerciales para la expresión, los promotores más eficientes, los marcadores seleccionables, la señal de secreción, los métodos usados en las transformaciones genéticas y los patrones de glicosilación que se presentan. Se brindan recomendaciones generales acerca de los parámetros de bioprocesos como la composición del medio, el pH, la temperatura, la velocidad de aireación, la inducción y las estrategias de alimentación para alcanzar altos valores de productividad. Se presentan los resultados de las aplicaciones de Pichia pastoris en la producción de dos vacunas en Cuba, la vacuna contra la hepatitis B y la vacuna para el control de la garrapata(AU)

Pichia pastoris metylotrofic yeast (currently classified as Komagataella phaffii) is one of the most important yeast for the production of heterologous proteins. The work presents an analysis of the main characteristics that are marked in the production of recombinant proteins expressed in Pichia pastoris. It describes the strains available for the transformation and production of recombinant proteins expressed in P. pastoris, the main commercial vectors for expression, the most efficient promoters, selectable markers, the secretion signal, the methods used in genetic transformations and glycosylation patterns that occur. General recommendations are provided on bioprocess parameters such as media composition, pH, temperature, aeration velocity, induction, and feeding strategies to achieve high productivity values. The results of Pichia pastoris applications for the production of two vaccines in Cuba, the hepatitis B vaccine and the tick control vaccine are shown(AU)

Genetically-engineered "all-in-one" vaccine platform for cancer immunotherapy

An essential step for cancer vaccination is to break the immunosuppression and elicit a tumor-specific immunity. A major hurdle against cancer therapeutic vaccination is the insufficient immune stimulation of the cancer vaccines and lack of a safe and efficient adjuvant for human use. We discovered a novel cancer immunostimulant, trichosanthin (TCS), that is a clinically used protein drug in China, and developed a well-adaptable protein-engineering method for making recombinant protein vaccines by fusion of an antigenic peptide, TCS, and a cell-penetrating peptide (CPP), termed an "all-in-one" vaccine, for transcutaneous cancer immunization. The TCS adjuvant effect on antigen presentation was investigated and the antitumor immunity of the vaccines was investigated using the different tumor models. The vaccines were prepared

Advance in glycosyltransferases, the important bioparts for production of diversified ginsenosides / 中国天然药物

Ginsenosides are a series of glycosylated triterpenoids predominantly originated from Panax species with multiple pharmacological activities such as anti-aging, mediatory effect on the immune system and the nervous system. During the biosynthesis of ginsenosides, glycosyltransferases play essential roles by transferring various sugar moieties to the sapogenins in contributing to form structure and bioactivity diversified ginsenosides, which makes them important bioparts for synthetic biology-based production of these valuable ginsenosides. In this review, we summarized the functional elucidated glycosyltransferases responsible for ginsenoside biosynthesis, the advance in the protein engineering of UDP-glycosyltransferases (UGTs) and their application with the aim to provide in-depth understanding on ginsenoside-related UGTs for the production of rare ginsenosides applying synthetic biology-based microbial cell factories in the future.

The insertion of bioactive peptides at the C-terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity

BACKGROUND: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. RESULTS: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a ß/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. CONCLUSION: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.

Recent advances in enzymatic production of alpha-keto acids / 生物工程学报

Alpha-keto acid is a bifunctional organic compound containing both carboxyl and ketone groups, and widely applied in the industries of food, pharmaceutical and cosmetics. Based on the demand of eco-friendly process, safety and sustainable development, production of α-keto acids by enzymatic conversion technology has been paid more and more attention. In this article, we review the status of α-keto acids biosynthesis from three aspects: enzymatic screening, enzymatic modification and optimization of enzymatic conversion conditions. Meanwhile, we also indicate future research directions for further improving α-keto acids production.

Research progresses in microbial 1,3-1,4-β-glucanase: protein engineering and industrial applications / 生物工程学报

1,3-1,4-β-glucanase (E.C.3.2.1.73) is an important industrial enzyme which cleave β-glucans into oligosaccharides through strictly cutting the β-1,4 glycosidic bonds in 3-O-substituted glucopyranose units. Microbial 1,3-1,4-β-glucanase belongs to retaining glycosyl hydrolases of family 16 with a jellyroll β-sandwich fold structure. The present paper reviews the industrial application and protein engineering of microbial β-glucanases in the last decades and forecasts the research prospects of microbial β-glucanases.

Protein engineering: from directed evolution to computational design / 生物工程学报

By constructing mutant libraries and utilizing high-throughput screening methods, directed evolution has emerged as the most popular strategy for protein design nowadays. In the past decade, taking advantages of computer performance and algorithms, computer-assisted protein design has rapidly developed and become a powerful method of protein engineering. Based on the simulation of protein structure and calculation of energy function, computational design can alter the substrate specificity and improve the thermostability of enzymes, as well as de novo design of artificial enzymes with expected functions. Recently, machine learning and other artificial intelligence technologies have also been applied to computational protein engineering, resulting in a series of remarkable applications. Along the lines of protein engineering, this paper reviews the progress and applications of computer-assisted protein design, and current trends and outlooks of the development.

Trend of hybrid enzyme design in the big data era / 生物工程学报

The high efficiency and stability of enzymes are the basis for industrial application. Hybrid enzyme suitable for industrial applications could be constructed by many molecular biology technologies including tandem fusion, domain insertion and post-translational protein conjugation. However, the low expression and activity of hybrid enzyme limit its application in industrial production, and multifunctional design of a specific protein domain has been becoming a new trend. With the advent of high-throughput sequencing, biologists are starting to wrestling with massive data sets. Besides, the concept of protein sectors and co-evolution provides novel insight into the relationship of protein structure and function. The residues-covariation of a protein sector displays preference, which imparts functional diversity to different enzymes in the same family. The covariation-residues in specific protein sectors can be located based on the analysis of massive data, and then these functional residues can be assembled in a new enzyme variant using the biotechnology of synthetic biology, thus completing the redesign of natural enzymes. This indicates a new stage of designing hybrid enzyme, as well as the new trend of protein design in the era of biological big data.

Recent advances in cell-free unnatural protein synthesis / 生物工程学报

Cell-free unnatural protein synthesis (CFUPS) as an emerging approach for protein engineering research, has been successfully applied in basic scientific studies (e.g., protein-protein interaction, protein-nucleic acid interaction) and industrial production (e.g., pharmaceutical proteins, protein materials). CFUPS can improve the engineering freedom and the process control by allowing free addition of genetic elements and chemicals as research purposes. It also can give protein novel characteristics, structures and functions, including post-translational modification of proteins, incorporation of reaction handles, synthesis of biophysical probes and polymeric protein. This article systematically reviews the unnatural amino acid incorporation methods (global suppression, stop codon suppression, frameshift suppression and unnatural base-pairs) and the application advances of unnatural amino acids in protein modifications, biophysical probes, enzyme engineering, biomaterials and biopharmaceutical protein production. The opportunities and challenges of the CFUPS system development and the wide application of industrialization are also illustrated with details.

Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector / 中华骨科杂志

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector. Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase chain reaction (PCR). hBMP2 gene was inserted into pTA2-T-easy and pSELECT-GFPzeo-MCS eukaryotic expression vector, and then transferred into competence DHSα cells. After screening, pSELEC-GFPzeo-hBMP2 was obtained and identified by sequence analysis. The recombinant vector pSELECT-GFP zeo-rhBMP2 was transfected into CHO cells. The successful trasfection was verified by fluorescence microscope in 48-72 hours. The RT-PCR and immunofluorescence was used to confirm the hBMP2 expression. Western Blotting was used to detect the secretion of hBMP2.Results A 1216 bp fragment was obtained by PCR, the same as expectant fragment. The recombined pSE-LECT-GFPzeo-hBMP2 eukaryotic expression vector was identified by restriction mapping and sequence analysis. The results were identical with that of reported hBMP2 sequence (Genebank NM-001200). The successful transfection was verified by fluorescence microscope in 48-72 hours. The stable expression in eukaryotic cells was confirmed by immunofluorescence and RT-PCR which showed an obvious band between 1000-2000 bp. Western Blotting identified the immunogenicity of recombinant human BMP2 with the molecular weight of about 17×103. Conclusion The pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was constructed successfully.

Protein Engineering of Microbial Lipases / 中国生物工程杂志

Microbial lipases are important industrial biocatalysts with the character of stereoselectivity,site selectivity and high catalytic activity with few side effects.They have been used widely in many industrial and agricultural fields.The technology of protein engineering has been successfully applied to improve the activity and stability of microbial lipases,which will raise the competitive capacity of microbial lipase preparations and enlarge theirs application fields.The strategies,the problems and the prospects of protein engineering technology which have been applied to modify the microbial lipases was surveied.

Site-saturation Mutagenesis Technology and Its Application in Protein Engineering / 中国生物工程杂志

Site-saturation mutagenesis is a newly-developed technology in protein engineering.By manipulating the encoding genes,it can rapidly obtain the mutants of desired proteins whose target residues are substituted by 19 other common amino acids.Site-saturation mutagenesis could serve not only as a powerful tool in protein engineering,but also as an important method in exploring the structure-function relationship of proteins.Several techniques were summarized to achieve site-saturation mutagenesis and introduce their application status in the protein engineering.The problem and promising future of its application were also discussed.

Effects of overexpression of stimulatory G proteins on the expression of the other G proteins in COS-1 cells

To investigate the interaction of stimulatory GTP binding protein (G(s)) pathways with others, we overexpressed wild type alpha subunit of G(s) (G(s) alpha), constitutively activated R201E G(s) alpha, and dominant negative G226A G(s) alpha in COS-1 cells by transfection with DEAE-dextran, respectively, The expression of various G proteins in the transfected cells was analyzed after 72 h by quantitative Western blots, and cAMP production by stimulation with isoproterenol and forskolin was quantitated using cAMP binding proteins, The expression of Gs alpha increased about 5-fold in the transfected cells, with concomitant increase in the small forms. However, there was no significant alteration the in the level of the alpha subunit of inhibitory G protein (G(i)) and G(q), and the beta subunits of G proteins. The cAMP level without stimulation increased in the cells transfected with G(s) alpha regardless to the type of mutation, Treatment with either isoproterenol or forskolin resulted in comparable increase of the cAMP level in all the transfected cells, though the ratio to its respective basal level was smaller in the G(s) alpha-transfected cells, From this experiment, we found that the expression of the other G proteins and the signaling pathway producing cAMP did not change significantly by transiently expressing wild type, constitutively activated type, and dominant negative type of G(s) alpha. Analysis of the effects of long-term expression of Gs alpha would contribute to better understanding on how the G(s) alpha signaling system interacts with other signaling pathways and how it adapts to the changed environments.

Research and Progress on Feed Phytase Reform by Protein Engineering / 微生物学通报

As a kind of additive in feed of monogastric animals, the application of natural phytase is limited due to its disadvantages. In this paper, the strategies of phytse reform was introduced. Furthermore, the research and progress on protein engineering of feed phytase was reviewed, including phytase over-expression, phytase thermostability, catalytic efficiency and optimum pH.

Development of Novel Microbial Lipase Resources / 微生物学通报

Microbial lipase,one of important industrial biocatalysts,has been used widely in many industrial and agricultural fields.It is always the research focus to screen,mine and develop the microbial lipases with novel catalytic activity and high stability.This paper introduces briefly the pathways and methods to mine novel microbial lipase resources from six aspects,including extremophile,metagenome,genome database,protein engineering,immobilization,chemical modification,etc.