Enzymatic resynthesis of the "reactive site" bond in the modified aprotinin derivatives [seco-15/16]aprotinin and [Di-seco-15/16,39/40]aprotinin

On incubation of [di-seco-15/16,39/40]aprotinin with human plasmin, porcine pancreatic kallikrein or bovine or porcine trypsin in neutral or slightly alkaline solutions [seco-39/40]aprotinin is slowly formed with enzymatic resynthesis of the reactive-site bond 15/16. With chymotrypsin, however, further degradation of [di-seco-15/16,39/40]aprotinin takes place without enzymatic resynthesis. The apparent rate constants for the synthesis of [seco-39/40]aprotinin with kallikrein and trypsin have been determined and indicate that the bond-forming reaction is 10-200-fold slower with [di-seco-15/16,39/40]aprotinin than with [seco-15/16]aprotinin. The newly formed [seco-39/40]aprotinin has similar kinetic constants for the complexation with its cognate enzymes as aprotinin, indicating that any distortion of the secondary binding region due to cleavage of the Arg39-Ala40 bond does not seriously influence binding and affinities.     

Synthesis, cloning and expression of recombinant aprotinin

Synthetic DNA fragments containing the coding sequence for the serine proteinase inhibitor aprotinin, also known as bovine pancreatic trypsin inhibitor (BPTI) a Kunitz type inhibitor were fused to form a synthetic aprotinin gene by the method of Khorana and cloned into E. coli. The synthetic gene is characterized by the presence of certain restriction sites. These restriction sites are unique within the used cloning system. Therefore, a great number of modifications can be achieved easily by exchange of appropriate restriction fragments. Using this method the variant [Glu52]aprotinin was obtained starting from the aprotinin gene. Both genes were successfully expressed in E. coli as fusion proteins with beta-galactosidase using vector pUR 278. No translation products could be detected in four other expression system (pUR 108, pDR 540, pKK 223-3 and pUC 8). [Glu52]aprotinin was purified and renatured after cyanogen bromide cleavage of the fusion protein. This recombinant [Glu52]aprotinin shows exactly the same trypsin-inhibitory profile as natural aprotinin.     

Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.     

Preparation of chemically 'mutated' aprotinin homologues by semisynthesis. P1 substitutions change inhibitory specificity

The semisynthesis of homologues of aprotinin (BPTI) is described. The P1 amino acid residue of these homologues was substituted by other amino acids using peptide synthetic methods. The reactive-site-modified inhibitor (with the Lys15-Ala16 peptide bond hydrolyzed) was used as starting material. All carboxyl groups of the modified inhibitor were esterified with methanol, then the Lys15 methyl ester group was hydrolyzed selectively. Afterwards, Lys15 itself was split off. A new amino acid residue was incorporated by using water-soluble carbodiimide combined with an acylation catalyst. tert-Butyl-ester-protected amino acids were used for reinsertion. The method was tested by re-insertion of Lys15 to reconstitute the original inhibitor. Thirteen BPTI homologues with coded (Lys, Glu, Gly, Ala, Val, Ile, Leu) or uncoded amino acids (Abu, Ape, aIle, Ahx, tLeu, Neo) in position 15 were synthesized and the specificity of the inhibitors investigated. Amongst these, [Val15]BPTI was shown to be an excellent inhibitor for human polymorphonuclear leukocyte elastase having a complex dissociation constant of 0.11 nM. This inhibitor showed no detectable affinity to bovine pancreatic trypsin.     

High-level secretion of biologically active aprotinin from the yeastPichia pastoris

Summary A synthetic gene encoding aprotinin (bovine pancreatic trypsin, inhibitor) was fused to theSaccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site.Pichia pastoris strains were developed to'express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion.P. pastoris containing a single copy of the vector secreed approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipoten with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.     

Influence of plasma proteinase inhibitors and aprotinin on trypsin-induced bradykinin release in vitro in man

The consumption of kininogen (measured as kinin-releasable material) was studied in an experimental model in vitro. Analyses were made following the addition of increasing amounts of human cationic trypsin to human serum and plasma. The consumption of kininogen was correlated with the degree of saturation of the plasma proteinase inhibitors alpha 2-macroglobulin (alpha 2-M) and alpha 1-proteinase inhibitor (alpha 1-PI) with trypsin in the presence and absence of aprotinin (Trasylol). The level of kininogen fell dramatically when alpha 2-M was saturated to 70% in spite of 90% free alpha 1-PI. Trypsin-alpha 2-M complexes had no effect on kininogen levels. 60 mumol/l of aprotinin, i.e. approximately 3 X 10(6) KIU/l, blocked only 60% of the trypsin-induced kininogen consumption in serum, while 15 mumol/l of aprotinin blocked 100% of this consumption in plasma. With increasing concentration of aprotinin in serum, a decreasing consumption of alpha 2-M and especially of alpha 1-PI was observed on the addition of trypsin. The high aprotinin concentration needed to block trypsin-induced kininogen cleavage in human serum or plasma may explain the poor clinical effect of aprotinin to date in human acute pancreatitis.     

Recovery of aprotinin from insulin industrial process effluent by affinity adsorption

Aprotinin is a protease inhibitor found in bovine organs and used as a valuable human therapeutic compound. In this work, a process for the recovery of aprotinin from insulin industrial process effluent via affinity adsorption on immobilized trypsin and chymotrypsin was developed. First, process conditions were set as a result of a study of the effects of pH and ionic strength on pure aprotinin adsorption and desorption utilizing an experimental design methodology. The best conditions obtained with immobilized trypsin as the ligand were adsorption at 0.018 M NaCl and pH 8.7 and desorption at 0.018 M NaCl and pH 2.1. For immobilized chymotrypsin, the best conditions were adsorption at 0.582 M NaCl and pH 8.0 and desorption at 0.582 M NaCl and pH 2.1. Recovery of the inhibitor from the effluent was carried out utilizing a two-step process: trypsin-agarose adsorption followed by chymotrypsin-agarose adsorption. Analysis of the chromatographic fractions by trypsin and chymotrypsin inhibition and capillary electrophoresis assays strongly suggested that the recovered inhibitor is aprotinin.     

Commercial production of aprotinin in transgenic maize seeds

The development of genetic transformation technology for plants has stimulated an interest in using transgenic plants as a novel manufacturing system for producing different classes of proteins of industrial and pharmaceutical value. In this regard, we report the generation and characterization of transgenic maize lines producing recombinant aprotinin. The transgenic aprotinin lines recovered were transformed with the aprotinin gene using the bar gene as a selectable marker. The bar and aprotinin genes were introduced into immature maize embryos via particle bombardment. Aprotinin gene expression was driven by the maize ubiquitin promoter and protein accumulation was targeted to the extracellular matrix. One line that showed a high level of aprotinin expression was characterized in detail. The protein accumulates primarily in the embryo of the seed. Southern blot analysis showed that the line had at least 20 copies of the bar and aprotinin genes. Further genetic analysis revealed that numerous plants derived from this transgenic line had a large range of levels of expression of the aprotinin gene (0–0.069%) of water-soluble protein in T₂ seeds. One plant lineage that showed stable expression after 4 selfing generations was recovered from the parental transgenic line. This line showed an accumulation of the protein in seeds that was comparable to the best T₂ lines, and the recombinant aprotinin could be effectively recovered and purified from seeds. Biochemical analysis of the purified aprotinin from seeds revealed that the recombinant aprotinin had the same molecular weight, N-terminal amino acid sequence, isoelectric point, and trypsin inhibition activity as native aprotinin. The demonstration that the recombinant aprotinin protein purified from transgenic maize seeds has biochemical and functional properties identical to its native counterpart provides a proof-of-concept example for producing new generation products for the pharmaceutical industry.     

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of ∼7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 μg porcine aprotinin inhibited 6 μg trypsin whereas 1 μg commercial soybean inhibitor inhibited only 1 μg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.     

Effect of covalent binding of aprotinin with a polysaccharide carrier on its inhibition of kallikrein from human plasma, porcine pancreatic kallikrein and trypsin

The inhibition of trypsin, human blood plasma kallikrein and porcine pancreatic kallikrein by aprotinin (native and immobilized on carboxymethyl ester of dextran) was investigated. The experimental values of Ki of native and immobilized aprotinin--enzyme complexes are equal to 0.037 and 0.045 nM for trypsin, 0.38 and 112.3 nM for pancreatic kallikrein and 34.4 and 454.5 nM for plasma kallikrein with N alpha-benzoyl-L-arginine ethyl ester as substrate, and to 82.6 and 231.7 nM for plasma kallikrein with a natural substrate--kininogen. These data suggest that covalent binding of aprotinin to the water-soluble polysaccharide carrier does not interfere with its interaction with trypsin, whereas the inhibition of kallikreins decreases, especially that of pancreatic kallikrein. The experimental results indicate the marked differences in the structure of the binding site of the active center (or its environment) of plasma and pancreatic kallikreins, on one hand, and trypsin, on the other, as well as the differences between the plasma and pancreatic kallikreins. A high requirement of kallikreins to the maintenance of the native conformation of aprotinin during immobilization is postulated.     

The urinary excretion of epidermal growth factor in the rat is reduced by aprotinin, a proteinase inhibitor.

The present study on the rat shows that i.v. administration of the proteinase inhibitor aprotinin reduces the urinary output of immunoreactive epidermal growth factor (EGF) while the amount of immunoreactive EGF in the kidneys is increased. This indicates that the EGF-precursor in the rat kidney in vivo is processed by an aprotinin inhibitable proteinase. EGF is produced in the kidneys as a precursor with a molecular weight of approximately 130 kDa. In rat urine, nanomolar amounts of 6 kDa EGF are excreted per 24 h together with small amounts of high molecular weight forms of EGF. During i.v. administration of aprotinin the median urinary output of immunoreactive EGF is reduced to 15% of the excretion of control rats (23 pmol/2 h versus 157 pmol/2 h, P less than 0.001). Especially the excretion of 6 kDa EGF is reduced (median excretion 12 pmol/2 h versus 134 pmol/2 h, P less than 0.001). The amount of immunoreactive EGF in the kidney tissue is increased after aprotinin administration (median amount 0.11 pmol EGF/mg protein versus less than 0.04 pmol EGF/mg protein, P less than 0.001). Neither the creatinine clearance, the total urinary protein output, nor the volume of urine produced was affected by aprotinin.     

Potent trypsin-resistant hGH-RH analogues.

A series of analogues of hGH-RH-(1-29)-NH2 designed to have metabolic stability has been synthesized. Standard Boc-SPPS was employed, modified to permit the guanidinylation of amino side-chains after chain assembly but before release from the resin. [Dat1, Har(11, 12, 20, 21, 29), Ala15, Nle27, Asp28]-, [Dat1, Har(11, 20, 29), Orn12, Ala15, Nle27, Asp28]-, and [Dat1, Gap(11,12, 21, 29), Ala15, Har20, Nle27, Asp28]-hGH-RH-(1-29)-NH2 were completely resistant to trypsin and about 50 times as potent as hGH-RH-(1-29)-NH2 itself when injected subcutaneously in rats. These peptides are candidates for clinical application in the therapy of GH deficiency.     

Synthesis, biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.     

Recombinant aprotinin produced in transgenic corn seed: extraction and purification studies

Expression in transgenic plants is potentially one of the most economical systems for large-scale production of valuable peptide and protein products. However, the downstream processing of recombinant proteins produced in plants has not been extensively studied. In this work, we studied the extraction and purification of recombinant aprotinin, a protease inhibitor used as a therapeutic compound, produced in transgenic corn seed. Conditions for extraction from transgenic corn meal that maximize aprotinin concentration and its fraction of the total soluble protein in the extract were found: pH 3.0 and 200 mM NaCl. Aprotinin, together with a native corn trypsin inhibitor (CTI), was captured using a tryspin-agarose column. These two inhibitors were separated using an agarose-IDA-Cu2+ column that proved to efficiently absorb the CTI while the recombinant aprotinin was collected in the flowthrough with purity of at least 79%. The high purity of the recombinant aprotinin was verified by SDS-PAGE and N-terminal sequencing. The overall recombinant aprotinin recovery yield and purification factor were 49% and 280, respectively. Because CTI was also purified, the recovery and purification process studied has the advantage of possible CTI co-production. Finally, the work presented here introduces additional information on the recovery and purification of recombinant proteins produced in plants and corroborates with past research on the potential use of plants as biorreactors.     

Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G.

The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor. Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P'2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity. Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10(-8) M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.     

Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography

Plants have attracted interest as hosts for protein expression because of the promise of a large production capacity and a low production cost. However, recovery costs remain a challenge as illustrated for recovery of recombinant aprotinin, a trypsin inhibitor, with removal of native corn trypsin inhibitor from transgenic corn (Azzoni et al. in Biotechnol Bioeng 80:268–276, 2002). When expression is targeted to corn grain fractions, dry milling can separate germ and endosperm fractions. Hence, only the product-containing fraction needs to be extracted, reducing the cost of extraction and the impurity level of the extract. Selective extraction conditions can reduce impurity levels to the point that low-cost adsorbents can result in relatively high purity levels. In this work, we attempted to achieve comparable purity with these lower cost methods. We replaced whole grain extraction and purification of recombinant aprotinin with sequential trypsin affinity and IMAC steps with an alternative of germ fraction extraction and purification with ion exchange and hydrophobic interaction chromatography (HIC). Using germ extraction at acidic pH supplemented with heat precipitation to remove additional host proteins resulted in a higher specific activity feed to the chromatographic steps. The cation exchange step provided 7.6× purification with 76.4% yield and no sodium dodecyl sulfate–polyacrylamide gel electrophoresis detectable native corn trypsin inhibitor. After the HIC step (2.7× step purification with 44.0% yield), the final product had a specific activity that was 75.3% of that of the affinity-purified aprotinin.     

Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor.

In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single-disulfide intermediate [30-51] plays a key role. We have investigated a recombinant analog of [30-51] using a 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51]Ala, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, [30-51]Ala consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-51], named P alpha P beta, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30-51]Ala are structurally independent. The partially folded structure of [30-51]Ala explains many of the properties of authentic [30-51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30-51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native-like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains.     

Prey-mediated effects of the protease inhibitor aprotinin on the predatory carabid beetle Nebria brevicollis

To investigate the potential non-target impacts of transgenic pest-resistant plants, prey-mediated impacts of a protease inhibitor (PI) on the predatory carabid, Nebria brevicollis, were investigated. The PI used was aprotinin, a serine PI of mammalian origin with insecticidal properties when incorporated in artificial diet or expressed in transgenic plants. Field-collected N. brevicollis adults, kept at 23 °C, 16:8 L:D, were fed, over their pre-aestivation activity period of 24 days, with Helicoverpa armigera larvae reared on an artificial diet containing 0.5% (w:w, fresh mass) aprotinin. These larvae contained 22.62 μg aprotinin/g insect. Control prey was reared on diet without aprotinin. Beetle survival and body mass were unaffected by prey type. Beetles consuming PI-fed prey lost significantly more mass than the control beetles during two periods of mass loss, but gained significantly more mass during the final period of mass gain. This was not due to differences in amounts of prey supplied or consumed. The final mass gain coincided with increased consumption of PI-prey. Female beetles were significantly heavier than males, but we found no consistent gender-based differences in response to PI-prey. At the end of the experiment, body mass of all beetles was similar to field-collected ones (approximately 55 mg). All experimental beetles had significantly lower activities of digestive cysteine proteases and the serine proteases chymotrypsin and trypsin than field-collected ones. Beetles consuming PI-fed prey had significantly lower levels of trypsin and higher levels of chymotrypsin and elastase than the control beetles.     

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.     

Carbohydrate-containing derivatives of the trypsin-kallikrein inhibitor aprotinin from bovine organs. II. Inhibitor coupled to the (carboxymethyl)dextran derivatives of D-galactose

The trypsin-kallikrein inhibitor aprotinin was coupled to (carboxymethyl)dextran derivatives of D-galactose. The conjugates contained 14 and 38 D-galactose residues/mol of protein, respectively. The apparent dissociation constants Ki of the complexes between trypsin and modified aprotinins proved to be one order of magnitude higher than the respective values for native aprotinin. The distribution of the modified aprotinins in rat organs after endocardial injection has been studied. The conjugates of aprotinin with (carboxymethyl)dextran derivatives of D-galactose were characterized by decreased clearance rates; they accumulated in the active form in liver. The accumulation was 2.5-10 times higher than native aprotinin for the time of observation (5 min-2 h).