Allelic variants of human cytochrome P450 1A1 (CYP1A1): effect of T461N and I462V substitutions on steroid hydroxylase specificity.

Steroid hydroxylation specificities were determined for the wild-type and the two allelic variants of the polymorphic human cytochrome P450 1A1 (CYP1A1) that were associated with amino acid exchanges near the active site of the enzyme. All three variants were expressed in insect cells using recombinant baculoviruses. Each variant protein was spectrally and enzymatically active, as judged by the ability of the prepared microsomes to catalyse O-dealkylation of ethoxyresorufin and pentoxyresorufin in cumene hydroperoxide-mediated reactions. With progesterone and testosterone as substrate, all variants of CYP1A1 exhibited high, but different steroid hydroxylation activities (8-40 pmol hydroxysteroid/min/pmol CYP1A1, i.e. approximately 800-4000 pmol/min/mg microsomal protein). All three variants exclusively catalysed 6beta-hydroxylation of both steroids. In addition, towards progesterone as substrate, all variants also catalysed 16alpha-hydroxylations with approximately half of the rate of 6beta-hydroxylation activity. With progesterone as substrate for hydroxylation in 6beta position, CYP1A1 T461N had the lowest catalytic efficiency (Vmax/Km) followed by the CYP1A1 I462V variant and the wild-type enzyme. For 16alpha-hydroxylation of progesterone, the catalytic efficiencies of the three variants are not statistically significantly different. With testosterone as substrate the CYP1A1 1462V variant catalysed 6beta-hydroxylation with an efficiency considered not significantly different compared to the wild-type, although both the apparent Km and Vmax were significantly decreased. In contrast, the CYP1A1 T461N variant exhibited significantly decreased catalytic efficiencies compared to both the 1462V variant and the wild-type enzyme. These results indicate that all three naturally occurring allelic variants of human CYP1A1 hydroxylate steroid hormones with varying efficiencies in a stereo- and regioselective manner, whereby the CYP1A1 T461N variant exhibited the lowest catalytic efficiency.

Production of human interleukin-8 expressed in Escherichia coli: from a laboratory scale for in vitro tests via a technical scale for animal studies to a process scale for a GMP-compatible production.

An Escherichia coli K 12 strain has been constructed for efficient expression of recombinant biologically active human IL-8 (Interleukin-8). The development of a fermentation and purification process from the laboratory scale (cells from 15 l fermentation broth) to a production scale (cells from 200 l fermentation broth) is described. Material obtained from the laboratory scale was used for initial in vitro studies and for the development of a biological assay. An upscale purification process starting from 80 l fermentation broth resulted in larger amounts of IL-8 needed for preclinical studies. This process includes a fully automated control of the initial affinity chromatography step. Finally, a production process which differed markedly from the small-scale processes was tailor-made for GMP conformity and economic considerations. It consists of a cell disruption step followed by two crossflow diafiltrations with different molecular weight cut offs and filtration rates, one cation exchange chromatography and a final dialysis step. In order to enhance the overall yield of biologically active IL-8, conditions for a resolubilisation of insoluble IL-8 present in the remaining pellet after cell disruption were worked out.

Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368.

A 4.3 kb EcoRI fragment carrying the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368, was cloned and completely sequenced. The gene codes for a protein of 410 amino acids and was expressed in Escherichia coli and B. subtilis. Protein extracts from the recombinant E. coli strains were able to hydroxylate corticosteroids in the 15 beta position when supplemented with an extract from a P450- mutant of B. megaterium ATCC 13368 as a source of megaredoxin and megaredoxin reductase. In contrast, 15 beta-hydroxylation was obtained in vitro and in vivo without the addition of external electron transfer proteins, when cytochrome P450meg was produced in B. subtilis 168. Protein extracts from nonrecombinant B. subtilis 168 could also support the in vitro hydroxylation by cytochrome P450meg produced in E. coli.

Plasminogen activators from the saliva of Desmodus rotundus (common vampire bat): unique fibrin specificity.

The saliva of D. rotundus contains at least four plasminogen activators (PAs) which all require fibrin as a cofactor. D. rotundus salivary PAs (DSPAs) exhibit a sequential array of structural motifs such as "Finger" (F), "EGF" (E), "Kringle" (K) and "Protease" (P) which was elucidated by cDNA cloning and sequencing. The respective domain organizations are: FEKP (DSPA alpha 1 and DSPA alpha 2), EKP (DSPA beta) and KP (DSPA gamma). In all four forms the plasmin-sensitive site of tPA is obliterated, indicating that they function as single-chain enzymes. DSPA alpha 1 differs from alpha 2 by amino acid substitutions found mainly in the F, E and K domain, 11% of the total sequence. DSPA beta and gamma, while being closely related to alpha 2, still exhibit 2 and 13 amino acid exchanges, respectively. These sequence heterogeneities, together with results of Southern blot hybridization experiments, strongly suggest that the four DSPA mRNA species originate from different genes. All four forms of DSPA have been expressed in animal cell culture and DSPA alpha 1 was chosen for a detailed pharmacological characterization. In vitro DSPA alpha 1 activity is enhanced 50,000-fold in the presence of fibrin, whereas the activity of single chain tPA is only enhanced 100-fold. At equally effective thrombolytic doses DSPA causes lower bleeding incidence in a rat mesenteric vein model and exhibits high potency, clot selectivity, and speed in the dissolution of fibrin embolized into the lung of anesthetized rats. In the copper coil-induced dog coronary heart infarction model, at doses that achieve patency at equal rates, reocclusion is significantly less frequent than with tPA. These results indicate that DSPA alpha 1 may be a safer and more efficacious thrombolytic agent than the PAs currently in clinical use.

Purification and properties of an Arthrobacter oxydans P52 carbamate hydrolase specific for the herbicide phenmedipham and nucleotide sequence of the corresponding gene.

Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.

The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression.

Complementary DNAs coding for four Desmodus rotundus salivary plasminogen activators (DSPAs) were isolated and characterized. The predicted amino acid sequences display structural features also found in tissue-type plasminogen activator. The largest forms (DSPA alpha 1 and -alpha 2) contain a signal peptide, a finger (F), an epidermal growth factor (EGF), a kringle, and a serine protease domain, whereas DSPA beta and -gamma lack the F and F-EGF domains, respectively. Additional differences between the four forms suggest that distinct genes code for the members of the DSPA family. Transfection of DSPA-encoding cDNAs, placed under the control of the simian virus 40 late promoter, into COS-1 cells resulted in the secretion of highly fibrin-dependent PAs.

Production of human adrenocorticotropin by cleavage of alkaline-phosphatase-derived fusion proteins containing repetitive recognition sequences for collagenases.

Recombinant plasmids coding for fusion proteins which consist of human adrenocorticotropin joined to N-terminal sequences of Escherichia coli alkaline phosphatase via collagenase-sensitive linkers were constructed and used for the production of these proteins by transformed E. coli cells. It was shown that repetitive linkers of the form -Gly-(Pro-Xaa-Gly)n-Pro- with n greater than or equal to 2 were cleaved by clostridiopeptidase A (Clostridium histolyticum) by orders of magnitude faster than corresponding nonrepetitive sequences (n = 1). The C-terminal cleavage product was Gly-Pro-adrenocorticotropin which could be converted to the authentic hormone by dipeptidyl peptidase IV. On the basis of these enzymatic reactions a procedure for the preparation of pure adrenocorticotropin was developed. Derivatives of alkaline phosphatase containing similar repetitive linker sequences were cleaved by clostridiopeptidase A as efficiently as the adrenocorticotropin fusion proteins.

Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli.

Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.

Properties of hybrid plasmids, consisting of parts of the mini-R1 factor Rsc11 and ColE1.

In vitro joining of the two small multicopy plasmids Rsc11 and ColE1 by a poly dAdT linker resulted in hybrid plasmids, which determine resistance to ampicillin and immunity to colicin E1. Isolation of the plasmid DNA from single colonies revealed that a variety of hybrid plasmids was formed. Cleavage of these plasmids with restriction endonucleases HinII, HindIII, EcoRI, SmaI and BamI and hybridization with ColE1 demonstrated that they contain different parts of the parent plasmids, Rsc11 and ColE1. Their copy number in the cell is between 6 and 15 per chromosome depending on the plasmid. None of these plasmids can replicate in polA mutants. Replication continues in the presence of chloramphenicol. This suggests that replication can only occur from the ColE1 origin and that the replication function of the Rsc11 part is lost. The hybrid plasmids are compatible with Rsc11 but not with ColE1. The comparison of the physical maps of these Rsc11--ColE1 hybrids with their functions allows a partial determination of the location of ampicillin resistance, replication and incompatibility on the Rsc11 genome.