Peptides analysis in digested edible bird's nest by HPLC-MS / 中国中药杂志

Edible bird's nest contains lots of glycoproteins. The glycosylation inhomogeneity for glycoprotein often results in wide range of molecular weight and the difficulty for protein separation and charaterization. In this paper, proteins in the edible bird's nest were extracted using multiple extractions, and then digested by PNgase F and trypsin. The digest mixture was separated with HPLC, and peptides were identified based on MS/MS data searching. The results indicated that the extracted proteins were amount to 79.7% of total protein in the edible bird's nest. More than 20 species of peptides in the digested mixture were identified. The sequences of these peptides showed similarity with some proteins from Swiss-prot. The research indicated that deglycosylation, tryptic digestion coupled with HPLC-MS/MS is a proper strategy for characterization of proteins in the edible bird's nest.

Inhibitory effect of folic acid/polyamide-amine as a miR-7 vector on the growth of glioma in mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore if folic acid/polyamide-amine (FA/PAMAM) enhances the therapeutic effect of miR-7gene therapy for glioma in vivo.</p><p><b>METHODS</b>The miR-7 gene was transfected into U251 glioma cells by FA/PAMAM. The efficiency of gene transfection was assessed by fluorescence microscopy. The miR-7 level was detect by quantitative RT-PCR. Intracranial glioma models were established in thymectomized mice, and FA/PAMAM nanoparticles were transplanted into the tumors in situ 3 days later. The animal survival was recorded and the gross tumor volume and degree of edema were observed by MRI. Apoptosis in the glioma cells and expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) were assessed by immunohistochemistry, and EGFR and AKT-2 protein expression was detected by Western blot assay.</p><p><b>RESULTS</b>Compared with the liposomes, the FA/PAMAM nanoparticles were more efficient to transfer miR-7 gene into U251 glioma cells, MRI showed that the tumor growth was much slower in the FA/PAMAM/miR-7 group, and the animal survival time was longer. The apoptosis rate was (5.3 ± 0.9)% in the control group, (11.4 ± 2.4)% in the liposome/miR-7 group, and (17.7 ± 3.7)% in the FA/PAMAM/miR-7 group. The immunohistochemical assay showed that the levels of PCNA, MMP-2 and MMP-9 protein in the FA/PAMAM/miR-7 group were (34.6 ± 5.4)%, (24.5 ± 4.1)%, (25.4 ± 5.1)%, respectively, significantly lower than those in the liposome/miR-7 group (49.3 ± 5.9)%, (31.7 ± 7.1)% and (39.4 ± 6.4)%, respectively, and those in the control group (57.3 ± 7.4)%, (45.4 ± 6.9)% and (55.1 ± 7.3)%, respectively (all P < 0.05). The expressions of EGFR and AKT-2 proteins were 1.09 ± 0.12 and 0.62 ± 0.10 in the control group, 0.63 ± 0.11 and 0.43 ± 0.07 in the liposome/miR-7 group, and significantly deceased (0.47 ± 0.09 and 0.31 ± 0.04, respectively) in the FA/PAMAM/miR-7 group (all P < 0.05).</p><p><b>CONCLUSION</b>Compared with the liposomes, FA/PAMAM can transfect miR-7 into glioma cells with a higher efficiency in vivo, makes a longer time of the drug action, and shows a certain inhibitory effect on the growth of glioma, therefore, might become a new drug targeting agent in gene therapy forglioma.</p>

Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry / 药学学报

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.

Comparison of adsorbent with varying arm length and ligand density for the purification of recombinant hepatitis B virus surface antigen / 生物工程学报

Novel hydrophobic absorbents were synthesized by immobilizing butyl derivative onto the highly cross-linked agarose beads manufatured in China, which are used as matrix. The effect of the spacer arm length (3C, 8C and 10C) and ligand density (from 13 to 45 micromol/mL) on the hydrophobicity were investigated using purified Hepatitis B surface antigen (HBsAg) expressed by CHO cell lines. Also considering the effects of salt concentration and pH on HBsAg recovery and purification factor, orthogonal experiment design method was used to evaluated the absorbents. The results showed the butyl-S absorbent with the spacer arm length of C8, the ligand density of 22 micromol/mL gel showed the best performance for the separation of HBsAg. Approximately 100% HBsAg recovery and 60 as purification fold were achieved by this media under the operating condition of pH 7.0 and 9% of salt concentrateion.

Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen / 生物工程学报

A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

Study on Photolysis of Vitamin B_(12) and New Method to Determine the Contents of Vitamin B_(12) in Fermentation Broth / 中国生物工程杂志

The photolytic behavior of deoxyadenosylcobalamin and methylcobalamin in water solution was investigated with high performance liquid chromatography. The results indicated that the photolytic cleavage rate increased with the light intensity, according to which a new method was developed to determine the concentration of vitamin B12 in fermentation broth. The samples were completely photolyzed after cell disruption. The content of vitamin B12 was obtained by determining the content of the hydroxycobalamin. The method shows many advantages, such as rapidness, high accuracy and low sample quantity needed, over traditions methods. The developed method may be used in the field of vitamin B12 fermentation.

Separation and purification of PEGylated rhG-CSF by two-step ion-exchange chromatography / 生物工程学报

In order to separate and purify the PEGylated recombinant human granulocyte stimulating factor (rhG-CSF) at large laboratory-scale level, a two-step ion-exchange chromatographic separation procedure was designed. Cation-exchange chromatography was applied first to separate PEGylated rhG-CSF from un-reacted rhG-CSF, followed by anion-exchange chromatography to dissolve individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the free PEG. The molecular weight of individual PEGylated rhG-CSF was determined by MALDI-TOF and SDS-PAGE. MALDI-TOF mass spectrometry revealed that the molecular weights of mono-, di- and tri-PEGylated rhG-CSF are 23.8 kD, 28.6kD and 33.8kD, respectively. Cell proliferation activity was detected by MTT assay using NFS-60 cell. The in vitro residual bioactivity of mono-, di- and tri-PEGylated rhG-CSF were 90%, 75% and 43% respectively, comparing with the un-conjugated rhG-CSF. These results indicated that the un-conjugated rhG-CSF and excess free PEG can be removed completely and the three conjugate species can be purified into homogeneity by the two consecutive ion-exchange chromatographic steps. The purification procedure is easy to scale-up, high in performance and recovery.

Separation of correctly refolded and mis-refolded consensus interferon by hydrophobic interaction chromatography / 生物工程学报

Hydrophobic interaction chromatography was used to separate correctly refolded and mis-refolded consensus interferon. The effects of ligand types, salt concentration, pH and flow rate were investigated. The best result could be obtained by using Butyl Sepharose 4 Fast Flow, 0.8 mol/L of ammonium sulfate, pH 8.3 and 90cm/h of linear flow rate. Reverse-phase HPLC analysis showed the purity of the pooled fraction was as high as 99.6%. The specific activity of purified consensus interferon was 2.3 x 10(9) IU/mg, The mass recovery of targeth protein was 36.7%.

Simultaneous purification of superoxide dismutase, catalase and hemoglobin from bovine erythrocyte lysate with PEG 600 as chaperon / 生物工程学报

Superoxide dismutase, catalase and hemoglobin were purified simultaneously from the same batch of bovine erythrocyte lysate. The process involves an initial anion exchange chromatography, followed by a hydrophobic interaction chromatography and gel filtration chromatography. 0.75% polyethylene glycol 600 was added as a purification chaperon before the anion exchange chromatography. The hemoglobin fraction passed through the ion exchange column without being retained. The superoxide dismutase and catalase were adsorbed by the column and were eluted separately during elution. The two eluted fractions containing crude superboxide dismutase and catalase were further purified with hydrophobic interaction chromatography and gel filtration chromatography in sequence. The specific activities of superoxide dismutase and catalase were 15932u/mg and 65918u/mg, respectively. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography were used to analyze the purity of the proteins. The purity of superoxide dismutase, catalase and hemoglobin were 77.6%, 81.9% and 99.9%, respectively. The total recoveries for superoxide dismutase, catalase and hemoglobin were 47.4%, 29.6% and 88.7%, respectively.

Analyses of Streptomyces coelicolor inner membrane proteome by multidimentional protein identification technology / 生物工程学报

Streptomyces coelicolor is the model species among streptomycetes. Until now, proteomic analyses of S. coelicolor have been conducted using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, few integral membrane proteins were identified due to the hydrophobic and low-abundance nature of these proteins. In this work, 154 possible inner membrane proteins from S. coelicolor were identified using high pH-proteinase K sample preparation method and multidimensional protein identification technology, among them 44 are integral membrane proteins containing at least one transmembrane domain, most peptides and their corresponding proteins were identified experimentally for the first time.

Polyethylene glycol-accompanied ion-exchange chromatography to purify recombinant hepatitis B virus surface antigen / 生物工程学报

The dissociation of virus-like particles of Hepatitis B surface antigen (HBsAg) during the adsorption-desorption on the solid-phase of chromatography is a main challenge for its purification. Herein, poly (ethylene glycol) (PEG) was applied as an additive during the purification of HBsAg from recombinant Chinese hamster ovary (CHO) cell culture to improve the HBsAg recovery and protect its structural assembly. The presence of 1% of PEG10000 in the mobile phase of ion-exchange chromatography (IEC) of DEAE-Sepharose FF column could increase the recovery of HBsAg from about 55% to 80%, with a similar purification (-fold) (about 12) compared with the absence of PEG. Importantly, glycosylated protein forms of HBsAg were reserved well by PEG-accompanied chromatography. Furthermore, size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analysis was performed on line to monitor the aggregates, particle size and molecular weight distribution of HBsAg. The results demonstrated that the HBsAg particle size and assembly are more homogenous after adding PEG in the mobile phase of IEC than no PEG added in the mobile phase.

Study on rhG-CSF modified with polyethylene glycol / 生物工程学报

Monomethoxy Polyethylene Glycol(mPEG20000) was activated by N-hydroxysuccinimede and analyzed by infrared spectrum and hydrolysis kinetics. In order to propose the optimized reaction conditions of mono-PEGylated rhG-CSF, orthogonal design of the experiment was investigated. Ion exchange chromatography was used to separate and purify PEGylated rhG-CSF from unPEGylated rhG-CSF. The purity of mono-PEGylated rhG-CSF was analyzed by high performance liquid chromatography (HPLC) to be 97%.

Influence of wall polymer and preparation process on the particle size and encapsulation of hemoglobin microcapsules / 生物工程学报

Methoxypoly (ethylene glycol)- block-poly (DL-lactide) (PELA) microcapsules containing bovine hemoglobin (BHb) were prepared by a W/O/W double emulsion-solvent diffusion process. The P50 and Hill coeffcient were 3466 Pa and 2.4 respectively, which were near to the natural bioactivity of bovine hemoglobin. The results suggested that polymer composition had significant influence on encapsulation efficiency and particle size of microcapsules. The encapsulation efficiency could reach 90% and the particle size 3 - 5 microm when the PELA copolymer containing MPEG 2000 block was used. The encapsulation efficiency and particle size increased with the concentration of PELA. Increasing the concentrations of NaCl in outer aqueous solution resulted in the increase of encapsulation efficiency and the decrease of particle size. As the concentration of stabilizer in outer aqueous solution increased in the range of 10 g/L to 20 g/L, the particle size reduced while encapsulation efficiency was increased, further increase of the stabilizer concentration would decrease encapsulation efficiency. Increasing of primary emulsion stirring rate was advantageous to the improvement of encapsulation efficiency though it had little influence on the particle size. The influence of re-emulsion stirring rate was complicated, which was not apparent in the case of large volume of re-emulsion solution. When the wall polymer and primary emulsion stirring rate were fixed, the encapsulation efficiency decreased as the particle size reduced.

Purification and fingerprinting development of Salvia miltiorrhiza Bunge by high-speed counter-current chromatography / 生物工程学报

In an attempt to apply high-speed counter-current chromatography HSCCC for TCM fingerprints, the separation and purification of the Chinese medicinal plant Salvia miltiorrhiza Bunge of different localities was realized using the technique. The equipments used include a HSCCC (TBE-300) of Shenzhen Tauto Biotech containing three connected preparative coils (diameter of tube = 2.6mm, total volume = 300mL) and a 20mL sample loop and a HPLC from Shimadzu of Japan with a Ultrasphere C18 column (150 x 4.6mm ID, 5microm) and a 20microL sample loop. Salvia miltiorrhiza Bunge samples from 3 locations were separated by HSCCC in a Step-wise elution program with solvent systems A (hexane:ethanol: water = 10:5.5:4.5) and B (hexane:ethanol: water = 10:7:3) at a speed of 900 r/min and a flow-rate of 2mL/min. All the 12 peak fractions were eluted within 13 hours. The contents of each component varied greatly in different samples, which confirmed previous observation that the locations and climates have a great impact on the TCM quality and also indicated a quality control system is necessary to safeguard the quality of the herb. The retention times of the 12 peak fractions from crude extracts of the samples were collected by HPLC and the absorption spectrums of the corresponding peaks were identified. The 12 components of the three crude samples were readily distinguishable and can be used as fingerprints of S. miltiorrhiza Bunge. The relative standard deviation of the HSCCC retention times was less than 3%, which satisfies the requirement of the national standard reference index. The components 7, 8 and 11 from the standards were identified to be crypototanshinone, tanshinone I and tanshinone II A respectively. This study demonstrates that if it is possible to apply HSCCC for TCM fingerprinting, especially with samples of high viscosity and highly absorptive components. The precision and the run time of fingerprinting can be further improved if larger volume and a temperature control system is used. With these and other improvements, HSCCC is expected to play an important role in TCM development.

Preparation of bovine lipid-free hemoglobin / 生物工程学报

A new method for preparation of Hb solution free of stromal lipid was described. Almost all the lipid in fresh hemolysate of bovine red blood was removed with hydrophobic interaction chromatography (HIC) in the presence of 2% PEG4000, 5% PEG4000, 2%PEG10000 or 5%PEG10000. With the adding of 5%PEG4000, the 80% of recovery of Hb in HIC was obtained and the maximum lipid absorbed by hydrophobic medium, butyl agarose -6B was 86.6 mg/mL. The activity (P50) of hemoglobin preparation was 3386.4 Pa torrs, and the Hill number was 2.54, which were near to that of the native red blood cells. The mechanism of removing lipid by HIC and the function of PEG in the process were discussed.

Progress in purification of human serum albumin / 生物工程学报

Human serum albumin(HSA) has been used clinically to treat a number of diseases with high dosage. Extremely pure puoduct is required in large-scale production. Plasma-derived HSA(pHSA) has long been produced by precipitation methods. Among them cold ethanol precipitation is dominant. However, chromatographic purification of HSA has been increasingly studied in the last few years. Application of chromatography, especially ionexchange, affinity, and size-exclusion, has opened a new area in the production of pHSA. A new challenge is the purification of recombinant HSA(rHSA). A successful approach involves STREAMLINE expanded bed adsorption to direct capture the target product from the fermentation broth. This novel process eliminates the need to separate the cells by centrifugation or membrane filtration. Ion exchange chromatography and hydrophobic chromatography play a central role in the purification scheme. Integration with other chromatographic techniques such as size-exclusion, metal chelate, and affinity gives improved purification results. Though innovative, the purification of rHSA still needs further improvement and optimization to increase product purity and process recovery.