Protein secondary structure determination by NMR. Application with recombinant human cyclophilin.

It is a unique trait of the NMR method for protein structure determination that a description of the polypeptide secondary structure can be obtained at an early stage and quite independently of the complete structure calculation. In this paper the procedures used for secondary structure determination are reviewed and placed in perspective relative to the other steps in a complete three-dimensional structure determination. As an illustration the identification of the regular secondary structure elements in human cyclophilin is described.

Effect of specific growth rates on productivity in continuous open and partial cell retention animal cell bioreactors.

A clonal derivative of a transfectant of the SP2/0 myeloma cell line producing a chimeric monoclonal antibody was cultivated in both continuous open and continuous partially-closed bioreactors. Using an open system for the determination of kinetic parameters, we showed that the production of this chimeric mAb was growth associated. As such, the volumetric productivity increased linearly with increasing dilution rate up to the maximum dilution rate. Three continuous cultivations employing partial cell retention were conducted. In agreement with mathematical predictions, the product titer and volumetric productivity were independent of the degree of cell retention when the total dilution was held constant. When cells were maintained at a low specific growth rate, the product titer was independent of dilution rate and the volumetric productivity increased with increasing dilution rate, again in agreement with mathematical predictions. Since the partially-closed bioreactor could be operated at dilution rates in excess of the maximum specific cellular growth rate, volumetric productivities were greater than those achievable in the open bioreactor. However, when cells were maintained at a high specific growth rate, cell accumulation was limited and product titers decreased at high dilution rates. Therefore, the volumetric productivity in this latter case did not increase at higher dilution rates.

Sanglifehrins A, B, C and D, novel cyclophilin-binding compounds isolated from Streptomyces sp. A92-308110. I. Taxonomy, fermentation, isolation and biological activity.

A novel class of macrolides for which the name sanglifehrins is proposed, has been discovered from actinomycete strains based on their high affinity binding for cyclophilin A (CypA), an immunophilin originally identified as a cytosolic protein binding cyclosporin A (CsA). The sanglifehrins were produced by Streptomyces sp. A92-308110. They were isolated and purified by extraction and several chromatographic, activity-guided steps. Sanglifehrins A and B exhibit a 10 to approximately 20 fold higher affinity for CypA than CsA, whereas the affinity of sanglifehrins C and D for CypA is comparable to that of CsA. Sanglifehrins exhibit a lower immunosuppressive activity than CsA when tested in the mixed lymphocyte reaction. Their in vitro activity indicates that they belong to a novel class of immunosuppressants.

Fractional gastrointestinal transit time: intra- and interindividual variation.

The intra- and interindividual variation in the gastrointestinal transit times was measured in 12 healthy adult test subjects using a scintigraphic method with 111In marked single unit tablets. Test conditions were semi-standardized. The variation within the same individual was as large as the variation between different subjects regarding the transit times through both the small and the large intestines. The small intestine transit time was 5 h (median) with an interquartile range of 4-7 h. The median transit time through the large intestine was 23.5 h (interquartile range 19.0-40.5 h). The coefficient of repeatability for the small intestinal and large intestinal transit time was 4 h and 33 h respectively. Our conclusion is that the 111In single unit capsule method can be used for measuring the fractional gastrointestinal transit times, but the intra- and interindividual variations are rather large and similar. From a radiation-hygienic point of view the 111In single unit capsule method is acceptable. The large inter- and intraindividual variation in gastrointestinal transit time might be an important factor in the absorption of certain drugs.

A novel approach for high level production of a recombinant human parathyroid hormone fragment in Escherichia coli.

We describe a novel approach to the production in E. coli of a peptide fragment derived from the human parathyroid hormone (hPTH). The first 38 amino acids of hPTH were fused at the amino terminus to a derivative of the bacteriophage T4-encoded gp55 protein, and were expressed in the E. coli cytoplasm in inclusion bodies at levels exceeding 50% of the total cell protein. Solubilization and subsequent incubation of the inclusion bodies in dilute hydrochloric acid facilitated the cleavage of an acid-labile aspartyl-prolyl peptide bond engineered into the fusion protein, thus releasing the hormone fragment directly from the inclusion body preparation. The amino-terminal prolyl-prolyl dipeptide-extension was subsequently removed by treatment with Lactococcus lactis dipeptidyl peptidase IV which was overexpressed in E. coli and purified to near homogeneity from the cytosol of the recombinant bacteria. In pilot-scale fermentations, more than 80 mg of pure hPTH(1-38) were isolated per liter of bacterial culture, with an overall yield of 35%. This process is suitable for scale-up, is cost effective, and by employing recombinant dipeptidyl peptidase IV, should be widely and directly applicable to the manufacturing of peptides of pharmaceutical interest.

Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture.

A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day(-1). The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day(-1). Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.

Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A. Identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as a key intermediate by enzymatic in vitro synthesis and by in vivo labeling techniques.

The biosynthesis of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation: Bmt, systematic name: 2(S)-amino-3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid) is proposed to involve two principal phases: the formation of a polyketide backbone and a subsequent transformation process to the final product. Here we report on the identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the end product of the first phase. The primary indication of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the key intermediate in the proposed biosynthetic route came from in vivo labeling studies with [1-13C,18O2]acetate, demonstrating retention of 18O in the 3-hydroxy group. Final identification of this intermediate in in vitro polyketide assays with enriched enzyme fractions of Tolypocladium niveum was achieved after development of highly sensitive and specific detection methods and by use of synthetic reference substances. Two additional methylated in vitro products could be detected and characterized as 4(R)-methyl-(E,E)-2,6-octadienoic acid and 4(R)-methyl-6(E)-octenoic acid by liquid chromatography-mass spectrometry analysis and comparison with synthetic reference samples. Their relevance for Bmt biosynthesis is discussed. Bmt polyketide synthase shows optimal activity at substrate concentrations of 200 microM acetyl-CoA, 150 microM malonyl-CoA, and 200 microM S-adenosylmethionine, around pH 7 and at 35 degrees C. Interestingly the Bmt backbone is released from the enzyme as a coenzyme A thioester, suggesting that subsequent transformation to Bmt takes place upon this activated intermediate.

Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor.

The interleukin-1 beta-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor. The full-length precursor form of the enzyme was expressed in Escherichia coli as insoluble inclusion bodies. Following solubilization and refolding of the 45-kDa protein, autoproteolytic conversion to a heterodimeric form containing 10- and 20-kDa subunits was observed. This enzyme had catalytic activity against both natural (interleukin-1 beta precursor) and synthetic peptide substrates. The inclusion of a specific inhibitor (SDZ 223-941) of the converting enzyme in the refolding mixture prevented proteolytic processing to the 10-/20-kDa form. Similarly, refolding under nonreducing conditions also prevented processing. Time course experiments showed that the 10-kDa subunit was released from the 45-kDa precursor before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role.