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81.
Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography 总被引:2,自引:0,他引:2
A. Zamboni I. Giuntini D. Gianesello F. Maddalena F. Rognoni D. Herbst 《Cytotechnology》1994,16(2):79-87
The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using125I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP
-Fetoprotein
- BSA
bovine serum albumine
- FCS
Fetal calf serum
- HRP
horseradish peroxidase
- OPD
o-phenylenediamine dihydrochloride
- I.P.
isoelectric point
- IEF
isoelectric focussing
- PBS
Phosphate buffered saline 相似文献
82.
Summary The statistical interpretation of the histogram representation of NMR spectra is described, leading to an estimation of the probability density function of the noise. The white-noise and Gaussian hypotheses are discussed, and a new estimator of the noise standard deviation is derived from the histogram strategy. The Bayesian approach to NMR signal detection is presented. This approach homogeneously combines prior knowledge, obtained from the histogram strategy, together with the posterior information resulting from the test of presence of a set of reference shapes in the neighbourhood of each data point. This scheme leads to a new strategy in the local detection of NMR signals in 2D and 3D spectra, which is illustrated by a complete peak-picking algorithm. 相似文献
83.
Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly 13C15N-labeled proteins. In-phase 13C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from 13C to 1H leads to E.COSY-type cross peaks, from which the 3JH
co coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin. 相似文献
84.
85.
A. M. Bajo L. G. Guijarro M. G. Juarranz P. Valenzuela P. Martinez J. C. Prieto 《Bioscience reports》1993,13(2):69-77
Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 M GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0±7.0 nM VIP, whereas the maximal activity (at 1 M VIP)corresponded to an increase of about 140% with respect to basal values (7.5±0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 M) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED50=1.8±1.4 nM)>VIP(ED50=25.0±7.0 nM)>PHI (ED50=725.0±127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of s and i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of125I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd=2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd=0.43 M, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium. 相似文献
86.
The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size. 相似文献
87.
Arthur M. Brown 《The Journal of membrane biology》1993,131(2):93-104
Summary Ion channels are signaling molecules and by them-selves perform no work. In this regard they are un like the usual membrane
enzyme effectors for G proteins. The pathways of G protein receptor, G protein and ion channels are, therefore, purely infor
mational in function. Because a single G protein may have several ion channels as effectors, the effects should be coordinated
and this seems to be the case. Inhibition of Ca2+ current and stimulation of K+ currents would have a greater impact than either alone. Additional flexibility is provided by spontane ous noise in the complexes
of G protein receptor, G protein, and ion channel. By having a non-zero setpoint, the range of control is extended and the
responses become bi-directional. 相似文献
88.
89.
Noboru Sueoka 《Journal of molecular evolution》1993,37(2):137-153
Using a general form of the directional mutation theory, this paper analyzes the effect of mutations in mutator genes on the
G + C content of DNA, the frequency of substitution mutations, and evolutionary changes (cumulative mutations) under various
degrees of selective constraints. Directional mutation theory predicts that when the mutational bias between A/T and G/C nucleotide
pairs is equilibrated with the base composition of a neutral set of DNA nucleotides, the mutation frequency per gene will
be much lower than the frequency immediately after the mutator mutation takes place. This prediction explains the wide variation
of the DNA G + C content among unicellular organisms and possibly also the wide intragenomic heterogeneity of third codon
positions for the genes of multicellular eukaryotes. The present analyses lead to several predictions that are not consistent
with a number of the frequently held assumptions in the field of molecular evolution, including belief in a constant rate
of evolution, symmetric branching of phylogenetic trees, the generality of higher mutation frequency for neutral sets of nucleotides,
the notion that mutator mutations are generally deleterious because of their high mutation rates, and teleological explanations
of DNA base composition.
Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992 相似文献
90.
Immobilization of phospholipase D from cabbage was studied with the aim of stabilizing the enzyme for its use in synthesis of phospholipids. It was shown that phospholipase D can be immobilized by adsorption to polymeric carriers containing long chain anchor groups such as octadecyl, octyl, or other alkyl residues. Starting from the crude enzyme, phospholipase D activity is preferentially bound (up to 100%) in competition with contaminating proteins. A prerequisite of high binding rates is the presence of calcium ions, which play a mediating role in the adsorption process. The maximum activity of the carrier-enzyme complexes depends upon the calcium concentration in the immobilization process and the carrier material (>/=10mM CaCl(2) with octadecyl-Si40, >/=40 mM CaCl(2) with octyl-sepharose and butyl-fractogel). Immobilization of phospholipase D to octyl-sepharose was shown to result in a distinctly increased storage stability and an enlarged pH-optimum range for the catalytic activity. Operational stability of different phospholipase D-carrier complexes was compared. (c) 1993 Wiley & Sons, Inc. 相似文献