Recently, although the passive immunization strategy based on antibody drugs has achieved certain results in the prevention and treatment of HIV, supplement of antibodies is necessary to maintain its long-term effective concentration. The key question is how to prolong the active time of antibodies. Due to the food safety and mucosal colonization stability of lactobacillus, the anti-HIV single-chain antibody fragments were inserted in its expression plasmid with homologous promotor and signal peptide, and plasmid was transformed into competent cells of WCFS1 strains by electroporation. Positive single colonies were screened out by erythromycin, and after culture, the anti-HIV single-chain antibodies were detected to be expressed in the bacterial supernatant and the surpernatant can inhibit the HIV pseudovirus. Compared with the wild-type WCFS1 strain, the growth capacity of the recombinant strain in vitro did not changed and the CFU of the bacterial solution could reach to10 9/ml after 24 hours’ fermentation; the colonization ability of the recombinant strain also did not change obviously, and the each square centimeter of monolayer epithelial cells could bind 10 5CFU colonies. Expressing anti-viral proteins via a live recombinant lactobacillus as host rectal mucosal colonizers gave a new proposal to prevent HIV homosexual transmission.
Heat shock protein 70 (HSP70), as a molecular chaperone and extensively studied protein in environmental toxicology. Previous studies had shown that GrHSP70 (the HSP70 gene of Gobiocypris rarus) was reported significantly up-regulated in liver by a concentration/time-dependent manner after pentachlorophenol (PCP) exposure. To explore the role of the promoter in regulating the expression of heat shock protein 70, Based on the HSP70 gene cDNA sequence information of Gobiocypris rarus obtained, the nucleotide sequence of the 5'flanking region of GrHSP70 was cloned using the chromosome walking technology. Biological information analysis showed that the length of 5' flanking region is 1 487bp before the predicted transcriptional start site (C). There were a series of putative transcription factor binding sites include ERE, Sp1, GRE, TBP, C/EBP, Oct-1, GATA-1, etc. Firefly luciferase (Fluc) and renilla luciferase (Rluc) reporter gene vectors with different deletions of GrHSP70 gene were constructed, after transient transfection into HeLa cells, the activity of dual-luciferase reporter gene was detected. It was confirmed that the obtained HSP70 gene promoter was active, and the core region of the GrHSP70 promoter was located between -1 487bp to -1 093bp from the transcriptional start site.Meanwhile, HeLa cells were successfully transfected the recombinant plasmid (pGL-HSP70 promoter-Luc +) and exposure to different concentrations of PCP, after 24 hours cultivation, the double fluorescence activity was examined. Compared with the control, the fluorescence activity increased significantly with the increase of PCP concentration. Indicate that PCP may induce GrHSP70 expression by activating the GrHSP70 promoter. However, the mechanism by which PCP regulates the synthesis of HSP70 in Gobiocypris rarus still needs further research.
Background: Alkaline protease is an important industrial protease with many applications, and its fermentative activity can’t meet the needs of industrial production.Objective: The aim is to improve the alkaline protease AprE production by screening promoters.Methods: Bacillus subtilis WB600, constructed in previous research, was served as the host strain for AprE production and four promoters (P1,P2,P-1-2,P-2-1) were screened to improve AprE production.Results: The results showed that four recombinant strains containing different promoters could successfully express alkaline protease AprE. After 48h of fermentation, the activity of AprE of P2 was 4 041U/ml, which was 1.23 fold of promoter P1.The highest enzyme activity of the double promoter recombinant B. subtilis WB600/P-2-1-aprE reached 6 125U/ml, which was 1.35 fold of the double promoter P-1-2.Conclusion: Collectively,an effective strategy for enhanced production of alkaline protease was provided.
Glucose oxidase (GOD) catalyzes the oxidation of glucose to hydrogen peroxide and gluconic acid, and has a broad commercial application. The recombinant Pichia pastoris G/GMH1 containing multi copies of the GOD gene along with the HAC1 expression cassette had been constructed in our previous works, and was used here as the starting strain to further introduce the chaperone disulfide isomerase gene PDI1 or/and malate dehydrogenase gene MDH1, and to investigate the effects of co-expression of the PDI1, MDH1 and HAC1 gene on the secretory production of GOD. No significant change was observed in cell growth of the recombinant strains after co-expression of these genes. The co-expression of HAC1 with PDI1 or MDH1 improved the extracellular specific GOD activity in the corresponding strain G/GMH1-MDH1 and G/GMH1-PDI1 by 10.4% and 17.3%, respectively, reaching 11 047.6U/g DCW and 11 731.9U/g DCW which, to the best of our knowledge, is the highest reported GOD activity of the recombinant P. pastoris cultured in shake flasks. The GOD production decreased slightly in the strain G/GMH1-PDI1-MDH1 with the co-expression of all three genes. Among these constructed strains, the transcription level of the GOD gene was only slightly increased in G/GMH1-PDI1-MDH1, which did not result in the increase in GOD production. The insertion of the additional PDI1 and MDH1 gene led to a varying increase in their relative transcriptional level, which demonstrated that these genes were transcribed and expressed successfully. The increased transcription of PDI1 and MDH1 gene in G/GMH1-PDI1 and G/GMH1-MDH1 might be related to the increase of GOD production. Although the GOD, HAC1, PDI1 and MDH1 gene were all up-regulated in G/GMH1-PDI1-MDH1, GOD production was not enhanced.
Objective: Uracil-DNA glycosylase (UDGase) is a tool enzyme widely used in qPCR, NGS and other related fields. Because of its application characters, only heat-labile UDGase has good application potential. There are only 2 species origins of heat-labile UDGase as developed tool enzymes, which are patent protected and expensive. Therefore developing new species origins of heat-labile UDGase is of urgent need.Methods: Based on previous studies and sequence analysis, it is speculated that Scophthalmus maximus has a heat-labile UDGase. It was confirmed that the liver homogenate exhibited UDGase activity. The gene of UDGase of Scophthalmus maximus, SmUDGase, was cloned from the liver homogenate. Recombinant expression of SmUDGase was achieved in E.coli, and the enzyme was purified and characterized.Results: equence alignments showed that the cloning of SmUDGase was successful. Gone through recombinant expression, purification with affinity and ion-exchange chromatography, the purified enzyme achieved a purity of 95%, a productive rate of 1.51mg/L, and a specific activity of 2 295.08U/mg. SmUDGase was heat-labile with a rapid decrease of activity above 40℃. For other enzymatic properties, such as pH range, metal ion dependency and sensitivity to the inhibitor, SmUDGase was consistent with the current commercial UDGase.Conclusion: The study successfully cloned and characterized a new species origin SmUDGase from Scophthalmus maximus. SmUDGase was heat-labile with other enzymatic properties close to current commercial UDGase. Recombinant expression and purification methods of the enzyme also were explored. The purified enzyme has basically reached the commercial production standards. These provided theoretical reference and technical reserve for development of tool enzymes of this kind.
In eukaryotic cells, protein transport between membrane structures such as endoplasmic reticulum, Golgi, and plasma membrane is mainly achieved via vesicle budding and fusion. The SNARE protein family plays a key role in mediating the fusion of vesicles with the target membrane structure. In the model organism Saccharomyces cerevisiae, systematic studies of SNARE proteins in the whole genome are still lacking. A set of plasmids for the labeling of all 24 SNAREs in S. cerevisiae with GFP are constructed. Most of the plasmids employ endogenous promoters for expression, with only a few mildly overexpressed, thus avoiding potential mislocalization caused by high overexpression. The subcellular localization of each SNARE is verified by co-localization with organelle markers. Results indicate that the localization of three SNAREs differs from those in existing literature: Bos1 localizes to early Golgi; and Snc1 and Bet1 localize to the late Golgi/early endosomes. In addition, Sec9 is detected at the bud tip and septum. This is the first time that the localization of Sec9 in vegetative cells has been observed. It is also the first comprehensive experimental evaluation of yeast SNARE subcellular localization. Furthermore, the constructed plasmids constitute a convenient tool set for future yeast cell biology studies.
Firstly,five expressing NADH-dependent glutamate dehydrogenase E.coli engineering bacterias are constructed,get a glutamate dehydrogenase AxyGDH from Amphibacillus xylanus.The optimum temperature is 60℃ and pH is 8.0,the specific enzyme activity is (903.1±24.6)U/mg,the half-life is 167h at this condition.Then, decide the enzyme production conditions of E.coli BL21(DE3)/pET-28a(+)-AxyGDH: Inducer IPTG concentration 0.7mmol/L,induction temperature 25℃.The optimized medium composition: Glycerol 11.3g/L, yeast powder 16.3g/L, MgSO4·7H2O 0.62g/L,NaCl 0.5g/L, Na2HPO4·12H2O 17.1g/L,KH2PO4 3g/L,NH4Cl 1.5g/L.At last,the fermentation enzyme activity of AxyGDH is (9 066±45)U/ml in the 10L tank fermentation when controlling the specific growth rate is 0.2h -1,which is 51.1 times than that of LB medium.It lays the foundation for glutamate dehydrogenase low cost production.
CRISPR-Cas9 system was first attempted to use in Angel industrial yeast - derived strain K-a, and HIS1 gene knockout was carried out. Because strain K-a is haploid, ura3 and sensitive to hygromycin B, the three key factors were designed as followings:Cas9 expressing vector YCplac33-Cas9 with URA3 marker, gRNA expressing vector pRS42H-gHIS1 with hph NT1 marker, and donor DNA fragment by PCR. By using lithium acetate method, pRS42H-gHIS1 and donor DNA was co-transformed into K-a (YCplac33-Cas9) competent cells and (CMG -URA+300μg/ml hygromycin B) plate was used for screening. The target transformants were proved to obtain and the ratio was 74.4% by phenotype screening and PCR product sequencing. So the industrial host platform utilizing CRISPR-Cas9 system was constructed and the simple and rapid gene manipulation protocol was preliminarily formed.
α-Glucosidase can cleave α-1,4 glucosidic linkages from the non-reducing end of oligosaccharide substrates to release glucoses, the enzyme also catalyzes transglucosylation, synthesizing α-1,6 glucosidic linkages. α-Glucosidase can be used in various fields such as isomaltooligosaccharide production, metabolic physiology research, disease prevention and treatment. Aspergillus nidulans-derived α-glucosidase has low enzymatic activity and degradation of protein in exogenous expression in Pichia pastoris. In order to improve the exogenous expression of Aspergillus nidulans-derived α-glucosidase in Pichia pastoris. A recombinant strain P.pastoris KM71/pPIC9K-AgbB/pPICZA-Mpr1 was constructed based on P.pastoris KM71/pPIC9K-AgbB which has been recombinantly expressed, finally optimized for 3.6L tank fermentation. The high-copy recombinant co-expressing strain obtained by screening showed that α-glucosidase transglucoside activity and protein content can reach 22.56U/ml and 0.52mg/ml in shake flask fermentation, respectively, which was 1.92 times and 1.27 times of the original strain in shake flask fermentation. After optimizing the temperature and methanol induction concentration of the recombinant co-expressing strain in 3.6L tank fermentation, the optimum fermentation conditions was 1% methanol concentration at 25℃.The enzyme activity and protein content of the co-expressed strains can reach 128.12U/ml and 1.81mg/ml, respectively, which was 1.96 times and 1.50 times of the original strain in 3.6L tank fermentation.
Compared to traditional small molecular drugs, biomacromolecular drugs have high specificity, and become one of the most promising areas in drug development in the 21st century. However, the natural barrier of cell membranes has prevented many potential intracellular drug targets from drug development. Cell-penetrating peptides (CPP) are a class of short peptides with membrane-permeating functions, could efficiently carry biomacromolecules such as nucleic acids and proteins into the cytoplasm through cell membranes and perform their functions. CPP have many advantages such as high efficiency and low toxicity on the transportation of biomacromolecular drugs. The mechanism of CPP mediated-cellular delivery of cargo can be divided into direct entry and endocytosis depending on whether energy is dependent. Direct entry could be divided into four models according to the way of pore formation : barrel model,toroid model,carpet model and inverted micelle model.endocytosis could be divided into micropinocytosis,clathrin-mediated endocytosis,caveolin-mediated endocytosis,heparan sulfate proteoglycans-mediated endocytosis, neuropilin-1-mediated endocytosis. The type, concentration of CPP, physicochemical properties and molecular size of cargo affected the process of CPP entry, and then determine its mechanism. To summarize the mechanism of CPP-mediated biomacromolecular entry, which provides a basis for the study of more efficient and targeted CPP and promote its application in biology and medicine research.
As important therapeutic drugs, human antibodies have been widely applied in clinical therapy in recent years. At present, the range of clinical application has gradually expanded from tumors, autoimmune diseases and chronic inflammation to cardiovascular diseases and infection, Compared with conventional therapy, the advantages of antibody therapy are specificity and high efficiency, which provides a new choice for the treatment of diseases. The entire structure of the human antibody is encoded by the human antibody gene, the human antibody gradually becomes the first choice for the development of therapeutic antibodies for avoiding the adverse reactions caused by the long-term application of the heterologous protein. The main preparation techniques and clinical application progress of therapeutic human antibodies are reviewed. At the same time, the disadvantages of human antibody preparation technology are also discussed to provide references and ideas for the development of human antibodies.
Epidermal growth factor receptor (EGFR) is an important transmembrane receptor with tyrosine kinase activity, which is abnormally expressed in a variety of malignant tumors and closely related to the proliferation, differentiation, metastasis and other life activities of tumor cells. At present, EGFR has been considered as a target for the treatment of tumors, and the drugs targeting on EGFR are mainly divided into two categories, one is monoclonal antibody drugs, and the other is small molecular kinase inhibitors. Small molecule inhibitors are prone to lead to EGFR mutation and drug resistance, thus affecting their clinical application. These mutations occur mainly near the ATP-binding site of the tyrosine kinase region, which are mainly deletion mutations on exon 19 and point mutations on exons 18 and 21.The types of drug-resistant mutations of EGFR and the ways in which they interact with small molecule inhibitors have been reviewed, with a view to providing references for the subsequent research and development of EGFR targeted drugs.
The sales of biologic drugs represented by monoclonal antibodies (mAbs) are expanding and showing a rising trend. The use of mAbs provides a new strategy for disease treatment.With the increase in the use of mAbs,higher requirements are placed on the quality of the product.As the level of mAbs expressed by the host cells continues to increase,the content of host cell proteins (HCP) also increases,and the upstream and downstream production processes are constantly faced challenge.The protein contained in HCP is extremely complicated.Although some HCP may be degraded, residual HCP may cause adverse reactions in the clinical use of the drug, thereby affecting the safety and effectiveness of the drug.Enzyme-linked immune sorbent assays (ELISAs) are important methods for HCP detection. ELISAs can quantitatively measure total HCP levels in drugs, but there are limitations.HCP testing of various analytical methods,including LC-MS/MS,is under development,which will provide more evidence for drug process development and validation.The advances in quality control and assays for HCP production in the production of mAbs hosted by the CHO (Chinese hamster ovary) cell line were discussed.
Silicon nanowire field-effect transistor(SiNW-FET) biosensor has been successfully used to detect many kinds of biomolecules.such as protein,nucleic acids,carbohydrates,etc.Compared with traditional detection methods,it has the advantages that specificity,label-free,real-time and so on.But Debye screening effect of semiconductor device seriously affect the detection sensitivity of SiNW-FET biosensor to biomolecules in blood sampal,especially for protein molecule. And to a great extent,it hindered the practical application of SiNW-FET biosensor. Therefore,effectively overcoming Debye screening effect is the key for the practical application of SiNW-FET biosensor.Now,the main ways of effectively overcome debye screening effect and achieve protein detection in blood sampal are dilution,desalination,protein purification,using biomolecule-permeable polymer layer,tailoring antibody and using aptamer replace antibody.
