To seek the reason of heterogeneity of recombinant HWTX-I (rHWTX-I) expressed in Pichia pastoris. We expressed HWTX-I gene of interest in Pichia pastoris GS115/HWTX-I. The heterogenous product expressed was separated, purified and identified by using Ion exchange HPLC, reverse HPLC, Tricine SDS-PAGE and MALDI-TOF Mass Spectrometry and then sequenced in both N-terminus and C-terminus. These results show that the heterogeneity of rHWTX-I results from the incomplete processing of signal peptide of N-terminus and the internal degradation of C-terminus. Biological activity assay shows that the activity of the heterogenous rHWTX-I only showed 30% activity compared with the native HWTX-I. The Solutions to how to avoid the heterogeneity are also discussed.
Animals ; Neurotoxins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Reptilian Proteins ; biosynthesis ; genetics ; Spider Venoms ; biosynthesis ; genetics

The Tagsk1 (Triticum asetium L. glycogen synthase kinase 1) gene derived from the genome of wheat salt-tolerance mutant RH8706-49 was cloned by PCR. The special primers designed according to full length cDNA sequence of Tagsk1 (AF525086). A binary expression vector pBI121-gsk1 containing Gus and Tagsk1 was constructed. And pBI121-gsk1 was introduced into the callus induced from mature embryos of salt-sensitive wheat H8706-34 and cv. China Spring by particle bombardment. The transformed callus were screened by Kanamycin and 0.5% NaCl. The salt-tolerance callus were obtained, which showed higher ability of salt-tolerance and could diffirentiate roots and buds on the medium containing 0.5% NaCl.
Adaptation, Physiological ; Biolistics ; DNA, Plant ; genetics ; Glycogen Synthase Kinases ; genetics ; Mutation ; Plant Proteins ; genetics ; Plants, Genetically Modified ; Salt-Tolerant Plants ; genetics ; Seeds ; genetics ; Sodium Chloride ; metabolism ; Transformation, Genetic ; Triticum ; enzymology ; genetics ; physiology

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.
DNA, Bacterial ; genetics ; Genetic Vectors ; Oryza ; genetics ; Plant Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Protein-Serine-Threonine Kinases ; genetics ; Rhizobium ; genetics ; Transformation, Genetic ; Xanthomonas

The key and crucial step of metabolic engineering during quinic acid biosynthesize using shikimic acid pathway is high expression of quinate 5-dehydrogenase. The gene qa-3 which code quinate 5-dehydrogenase from Neurospora crassa doesn't express in Escherichia coli. By contrast with codon usage in Escherichia coli, there are 27 rare codons in qa-3, including eight AGG/AGA (Arg) and nine GGG (Gly). Two AGG are joined together (called box R) and some GGG codons are relative concentrate (called box G). Along with the secondary structure of mRNA analysed in computer, the free energy of mRNA changes a lot from -374.3 kJ/mol to least -80.5 kJ/mol when some bases in the end of qa-3 were transformed, and moreover, the change of free energy is quite small when only some bases in the box G and box R transformed. After the change of rare codon and optimization of some bases in the end, qa-3 was expression in E. coli and also the enzyme activity of quinate 5-dehydrogenase can be surveyed accurately. All the work above benefit the further research on producing quinic acid engineering bacterium.
Alcohol Oxidoreductases ; biosynthesis ; genetics ; Base Sequence ; Codon ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Hydro-Lyases ; genetics ; Molecular Sequence Data ; Neurospora crassa ; enzymology ; genetics ; RNA, Messenger ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Shikimic Acid ; metabolism ; Transformation, Bacterial

The effect of dual promoters on recombinant protein production from bacterial phage based Bacillus subtilis expression system was investigated. Alpha amylase (from Bacillus amyloliquefaciens) and penicillin acylase (from Bacillus megaterium) were selected as the indicating enzymes. Both the promoterless genes and the promoter-bearing genes were isolated through PCR amplification with properly designed primers, and were inserted into plasmid pSG703 that contains the lacZ-cat expression cartridge. The lysogenic B. subtilis (phi105 MU331) was transformed with the resultant recombinant plasmids, and the heterologous genes were thereby integrated into the chromosommal DNA of B. subtilis via homologous recombination. The transformants were designated as B. subtilis AMY1, B. subtilis AMY2, B. subtilis PA1, and B. subtilis PA2, respectively. In the recombinant B. subtilis strains, the inserted sequences were located down stream of a strong phage promoter that could be activated by thermal induction. In B. subtilis AMY1 and B. subtilis PA1, transcription of the heterologous genes was only initiated by the phage promoter after heat shock, whereas in B. subtilis AMY2 and B. subtilis PA2, transcription of the heterologous genes was initiated by dual promoters, the phage promoter and the native promoter. The application of dual promoters increased the productivity of both enzymes, with 133% enhancement for alpha-amylase production and 113% enhancement for penicillin acylase production.
Bacillus Phages ; genetics ; metabolism ; Bacillus subtilis ; genetics ; metabolism ; Cloning, Molecular ; Penicillin Amidase ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Recombinant Proteins ; biosynthesis ; genetics ; Transformation, Bacterial ; alpha-Amylases ; biosynthesis ; genetics

To construct a safer and more efficient gene engineering Lactococcus Lactis for expressing phenylalaine ammonia lyase (PAL) which will be benefit for PKU therapy, pal cDNA of Parsly and synthesized sequence based on Lactococcus Lactis bias codons were recombined into two Lactococcus Lactis NICE systems. The activities of the expressed PAL were detected, and the effect of Lactococcus Lactis bias codons on the expression of exterior protein was analyzed. The results showed that the expression level of PAL was increased by using Lactococcus Lactis bias codons in both Lactococcus Lactis NICE systems. Through which several safer andmore efficient strains of the gene engineering Lactococcus Lactis were obtained.
Cloning, Molecular ; Codon ; genetics ; Genetic Vectors ; genetics ; Lactococcus lactis ; genetics ; metabolism ; Phenylalanine Ammonia-Lyase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; metabolism ; Transformation, Bacterial

Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.
Animals ; DnaB Helicases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Inteins ; genetics ; Natriuretic Peptide, Brain ; biosynthesis ; genetics ; Protein Engineering ; methods ; Protein Splicing ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.
Animals ; Bioreactors ; microbiology ; Fermentation ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; Macaca fascicularis ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacokinetics ; Recombinant Proteins ; Serum Albumin ; biosynthesis ; genetics

As a novel analytical technology, the research of Micro total analysis systems (micro-TAS) has been spreading rapidly. micro-TAS has been widely used to perform chemical and biochemical analysis. Microfluidic-based analytical system as micro-TAS's manily direction develops very fast in terms of it's reaction speed, reagent consumption, miniaturization, cost, and automation. After having proven the value of microfluidics for genetic, proteomic and cytomics analysis, this article also anticipates the development tendency of this technology in the biology medicine domain. It has demonstrated that a truly, easy-to-handle Microfluidic-based analytical device will be emerged in the future.
Humans ; Microarray Analysis ; instrumentation ; methods ; Microfluidic Analytical Techniques ; trends ; Microfluidics ; trends ; Technology Assessment, Biomedical

Cell transplantation is a promising technology in cancer therapy, however, immunological rejection is the major problem of cell transplantation. Based on the permselective property of microcapsule membrane, encapsulated cells can be immuno-protected. The normal physiological state and function expression of cells can be maintained so as to realize allo- or xenotransplantation. The microencapsulated cells grow in three dimensions, giving a more biologically representative in vivo model, which hints difference in characters of growth and metabolism compared to the monolayer cells. Therefore, characterization of growth and metabolism of microencapsulated recombinant CHO cells is essential for further large-scale culture. In present study, the effect of concentration of glutamine on the growth, metabolism and endostatin production of microencapsulated cells was investigated. In the experimental range of initial glutamine concentrations from 2.69mmol/L to 9.05mmol/L in the culture of microencapsulated recombinant CHO cells, the maximum density of active cells and multiplication ratios almost kept constant. The specific consumption rate of glucose increased with lower initial glutamine concentration (2.69mmol/L). When initial glutamine concentration was much higher (7.91mmol/L to approximately 9.05mmol/L), the specific consumption rates of both glucose and glutamine increased while the efficiencies of glucose and glutamine decreased. The highest efficiencies of glucose and glutamine utilization were observed with initial glutamine concentration of 4.97mmol/L. It was also demonstrated that glutamine had significant effect on the accumulation of endostatin. The accumulative concentration of endostain reached its peak of 546.36 ng/mL with the initial glutamine concentration of 4.97mmol/L.
Animals ; CHO Cells ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Cells, Immobilized ; Cricetinae ; Cricetulus ; Culture Media ; Endostatins ; biosynthesis ; genetics ; Genetic Engineering ; Glucose ; metabolism ; Glutamine ; pharmacology ; Recombination, Genetic