This book presents various approaches encompassing biological, biochemical and genetic aspects as well as recovery of the final end product by means of large scale production, with emphasis on fundamental explanations of the techniques and their practical success in protein production with mammalian cell cultures.The book therefore handles a broad spectrum of subjects relevant to protein production including virology, biochemistry, molecular genetics, cell biology, bioreactor technology, process simulation, validation, biophysics, hydrodynamics and legal aspects.
The mutual effects of P. gingivalis and several cystatin species has been investigated.After incubation with P gingivalis culture supernatant, cystatin S, cystatin C and chicken cystatin were truncated from a 14 kDa protein into a polypeptide of approximately 13 kDa.Amino acid sequence analysis of the truncated cystatin S polypeptide revealed that cystatin S was cleaved after Arg-8.All three types of truncated cystatins fully retained their inhibitory activity toward papain.Cystatin S and chicken cystatin partially inhibited proteolytic activity in the culture supernatant of P. gingivalis.Furthermore, cystatin S and chicken cystatin inhibited the growth of R gingivalis in culture to 50% at approximately 1 μΜ.
We have used a yeast one hybrid screen to search for factors interacting with a subsegment of the immunoglobulin heavy chain (IgH) intronic enhancer.The 51 bp enhancer segment harbored a so-called -box and an octamer site, known to bind helix-loop-helix transcription factors and Oct factors, respectively.Mammalian Oct-2A protein was also expressed in yeast, to select for transcription factors possibly cooperating with Oct-2.Six strongly interacting protein clones were selected from a peripheral blood lymphocyte library.These included a B cell-specific coactivator, termed Bob1, that directly binds to Oct-2 (Gstaiger et a/., 1995, Nature 373, 360 -362).Three further clones represent the helix-loop factors ITF-1 and ITF-2, another one the nucleolar protein nucleophosmin, or B23.Unexpectedly, the sixth clone with strong activity encoded the BZLF1 (= ZLF1, zta, ZEBRA, EB1) protein of Epstein-Barr virus (EBV).BZLF1 is a leucine zipper-related transcription factor and induces the switch from viral latency to lytic growth.We found that BZLF1 also activated transcription in transiently transfected mammalian cells via a consensus binding site located within the IgH intron enhancer.BZLF1 may thus influence immunoglobulin heavy chain expression in EBV-infected B lymphocytes.
A novel brain-type member of the fatty acid binding protein family (B-FABP) was heterologously expressed in Escherichia coli, either as inclusion bodies at 37 degrees C or in soluble form at 22 degrees C. Both B-FABP renatured from inclusion bodies and the solubly expressed protein could be purified to homogeneity by anion exchange chromatography and gel filtration in a functional conformation as they bound oleic acid with high affinity. None of the five cysteines of B-FABP was involved in disulphide bond formation. Isoelectric focusing revealed heterogeneity of the renatured protein but not of the solubly expressed protein. By Western blotting using affinity purified rabbit antibodies raised against the recombinant B-FABP it was demonstrated that in adult mice, B-FABP is predominantly expressed in the olfactory bulb.
Cathepsins B and L are thought to function extracellularly in pathological conditions. pH-Activity profiles of cathepsin B, measured in phosphate and acetate-Mes-Tris buffers of constant ionic strength, indicated that cathepsin B is sensitive to specific buffer ions, as previously reported for cathepsin L. In assessing the activity of these enzymes in vitro the influence of the buffer must therefore be taken into account. In Hank's balanced salt solution, a buffer modeling the extracellular fluid, the half-life of activated human liver cathepsin B at 37 degrees C is 245 +/- 11.3 s, at pH 7.2, and 857 +/- 50.1 s, at pH 6.8 (the peritumor pH), indicating that cathepsin B is markedly stable under these conditions. The stability was increased by the additional presence of proteins. Without immediate activation, however, the stabilities of both cathepsins B and L were markedly decreased, a large proportion of their activity being lost before it could be measured. Enzymes injected into the extracellular space in the unactivated state would therefore survive for only a very short time in their native conformation. It is proposed that the active site thiolate-imidazolium ion pair contributes substantially to the stability of cathepsins B and L to extracellular ionic conditions.
Novel angiotensin-I-converting enzyme (ACE) inhibitory activities were detected in synthetic peptides corresponding to sequences of beta-lactoglobulin and alpha-lactalbumin and which are known to possess opioid activity. Using hippuryl-histidyl-leucine as substrate, the tetrapeptides beta-lactorphin (Tyr-Leu-Leu-Phe), alpha-lactorphin (Tyr-Gly-Leu-Phe) and beta-lactotensin (His-Ile-Arg-Leu) were shown to have IC50 values of 171.8, 733.3 and 1153.2 microM, respectively. Related dipeptides also inhibited ACE, with Tyr-Leu being the most potent, having an IC50 value of 122.1 microM.
Recombinant DNA technology has enabled the large scale production of human hemoglobin in bacteria and yeast. This has opened up a way to produce a hemoglobin-based blood substitute which could replace conventional blood transfusion in some situations. Using our understanding of the structure-function relationships and evolutionary history of hemoglobin it has been possible to improve the oxygen transport properties of the molecule and solve a number of problems associated with the use of natural hemoglobin as a cell-free blood substitute.
Dehydration stress in the drought-tolerant resurrection plant Craterostigma plantagineum is accompanied by the accumulation of a large number of desiccation stress proteins (Dsp). One abundant class of these is represented by the dehydrin-related Dsp16 protein which contains 15 amino acid conserved lysine-rich repeats and a stretch of eight serine residues providing extremely hydrophilic characteristics. Recombinant Dsp16 from Craterostigma plantagineum has been cloned and expressed in Escherichia coli. The protein was purified and characterized regarding its physicochemical properties. Irrespective of successful crystallization experiments, dilute aqueous buffer solutions do not display a well-defined three-dimensional structure in terms of the canonical secondary structural elements. 1H-NMR (nuclear magnetic resonance) spectra in aqueous solution are characterized by a small chemical shift dispersion typical for an unfolded protein; however, the observed line-widths are not typical for a highly mobile random coil structure. Instead they indicate an equilibrium between conformational states with preferentially extended substructures. As a consequence of its loose structure, Dsp16 is extremely sensitive towards proteolysis unless its structure is stabilized by structure-making additives such as trifluoroethanol. Denaturants such as guanidinium chloride do not induce cooperative structural transitions. pH-dependent fluorescence changes reflect protonation/deprotonation rather than conformational changes. Sedimentation/diffusion experiments confirm the predicted molecular mass of 16 kDa. Due to the high serine/threonine content and its loose structure, Dsp16 is accessible to phosphorylation, supporting the idea that in situ the structurally relatively undefined protein may be involved in both water binding and phosphorylation.
A novel purification procedure was developed for pyruvate decarboxylase (PDC, E.C. 1.1.1.4) from the haploid yeast strain YSH 4.127-1A expressing only one (PDC1) of the three structural genes for PDC. The purified enzyme is homotetrameric with a molecular mass of about 240,000 whereas PDC from brewer's yeast is a dimer of dimers composed of subunits of different size (alpha 2 beta 2) with the same molecular mass as the tetramer. Despite these structural variations there are no significant differences in the kinetic behaviour of the two enzyme species. PDC purified from the haploid yeast mutants shows a sigmoid dependence of the reaction rate from the substrate concentration due to the substrate activation. In the presence of the substrate surrogate pyruvamide the shape of the v/S plot is transformed into a hyperbolic one. As expected, polyclonal antibodies react with both the enzyme from haploid yeast strain mutants and that from brewer's yeast.
A novel purification procedure was developed for pyruvate decarboxylase (PDC, E.C. 1.1.1.4) from the haploid yeast strain YSH 4.127-1A expressing only one (PDC1) of the three structural genes for PDC. The purified enzyme is homotetrameric with a molecular mass of about 240,000 whereas PDC from brewer's yeast is a dimer of dimers composed of subunits of different size (alpha 2 beta 2) with the same molecular mass as the tetramer. Despite these structural variations there are no significant differences in the kinetic behaviour of the two enzyme species. PDC purified from the haploid yeast mutants shows a sigmoid dependence of the reaction rate from the substrate concentration due to the substrate activation. In the presence of the substrate surrogate pyruvamide the shape of the v/S plot is transformed into a hyperbolic one. As expected, polyclonal antibodies react with both the enzyme from haploid yeast strain mutants and that from brewer's yeast.
The nuclease resistance of an oligonucleotide sequence that was phosphorothioate (PS)-modified in various positions and patterns was examined. We present a new ¿minimal' protection strategy for antisense oligonucleotides which is a combination of the end-capping technique and the protection of internal pyrimidine residues which are the major sites of endonuclease degradation. This strategy reduces the number of modifications needed to make a nuclease resistant oligonucleotide and therefore should minimize the non-sequence-specific effects that are frequently observed with uniformly modified oligonucleotides.
Insulin from a monotreme, the platypus (Ornithorhynchus anatinus), was isolated and the amino acid sequence determined.It differs from pig insulin at eleven amino acid sites, mainly on the surface of the monomer.Substitutions relative to pig insulin occur in the monomer-monomer interface, the dimer-dimer interface and the receptor binding region.The residues A5 Glu, A8 Lys and A13 Met have not been reported before in any insulin.Multiple sequence comparison studies reveal a relatively close relationship with the nearest group of relatives to the platypus, the mammals.The relationship of the platypus sequence to reptilian insulin sequences (and amphibian and avian insulin sequences in this case) is sufficiently close to support the observation that platypus has retained some ancient reptilian characteristics over the course of evolution.Model building the platypus insulin sequence on the structure of porcine insulin indicates that there may be some interesting differences.
In Arabidopsis mitochondria the nad2-gene consists of five exons (a-e) which are separated by three cis-splicing introns and one trans-splicing intron. Sequence analysis of the region upstream of exons a and b reveals an open reading frame encoding ribosomal protein S4 (rps4). In the second nad2 coding region (exons c-e) a pseudo tRNA(Tyr) sequence and a fragment of the plastid psbA gene are located upstream of the trans-spliced exon c. Primer extension analysis identifies RNA 5'-termini within the pseudo-tRNA(Tyr) confirming this sequence to be non-functional. Northern blot analysis suggests the rps4-gene to be cotranscribed with at least the first part of the nad2-gene. The rps4 and nad2 coding sequences as well as the first cis-intron and the trans-intron sequences of the nad2 gene are altered by RNA editing. RNA editing in the open reading frames improves in most instances conservation of the specified amino acids.
From a murine B-cell cDNA-library we have cloned a cDNA encoding the murine B-cell specific coactivator mBob1. The protein is the murine homologue to the recently described human coactivator Bob1 (hBob1), also referred to as OBF-1 or OCA-B. We have also characterized the genomic mBob1 clone. Analysis of its intron-exon structure has allowed identification of a C-terminal splice variant. mBob1 is B-cell restricted, and is found in all B-cell lines representing different stages of B-cell differentiation. mBob1 interacts with the octamer transcription factors Oct-1 and Oct-2 and stimulates transcription mediated by these factors.
The possible application of proteinase inhibitors in the support of anti-tumor chemotherapy requires profound knowledge of the proteinases involved in malignant processes. Therefore, the occurrence of cathepsins B, D, H, L and S and of gelatinases, urokinase plasminogen activator and stromelysins was studied in biopsies of aggressive human bone metastases, of low invading basal cell carcinomas, and in normal placenta as control, by activity measurements and zymographic techniques. Cathepsin B and L, as well as gelatinase B, were shown to be overexpressed in bone metastases, suggesting a function during the metastatic process. Subcellular fractionation allowed detection of differential sorting of cathepsin B and gelatinases in metastatic tissue and also in normal human placenta. Plasma membrane binding could be demonstrated for both cathepsin B and gelatinase B. Whereas cathepsin B is at least partially bound to plasma membranes via alpha 2-macroglobulin and its LRP/alpha 2-macroglobulin receptor, gelatinase B binds to plasma membranes by an unknown mechanism.
c-Jun and JunD are two closely related members of the Jun family of transcription factors which markedly differ in their biological functions. Whereas c-Jun behaves as a positive regulator of cell growth and may cause cell transformation when overexpressed, JunD antagonizes both of these effects. To better understand how the activities of c-Jun and JunD are controlled, we investigated how their stabilities within the cell are determined. We show that, in contrast to c-Jun which is degraded following multi ubiquitination, JunD is not efficiently ubiquitinated and exhibits a correspondingly longer half-life. Mutational analysis reveals that the determinant for the difference in ubiquitination resides in the NH2-terminal regions of the proteins which in c-Jun contains the delta-domain.
Discovery of the protein machinery of vesicle transport has followed the reconstitution of transport in a cell-free system, and established general mechanisms that underlie a broad variety of physiological processes, including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled release of hormones and neurotransmitters. Transport vesicles are formed as coat proteins assemble on membranes, are targetted by SNARE proteins, and fuse using a general mechanism involving SNAP proteins and the ATPase NSF.
