Production of spider silk proteins in tobacco and potato.

Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90% compared to their native models. Here, we demonstrate the accumulation of recombinant silk proteins, which are encoded by synthetic genes of 420-3,600 base pairs, up to a level of at least 2% of total soluble protein in the endoplasmic reticulum (ER) of tobacco and potato leaves and potato tubers, respectively. Using the present expression system, spider silk proteins up to 100 kDa could be detected in plant tissues. When produced in plants, the recombinant spidroins exhibit extreme heat stability-a property that is used to purify the spidroins by a simple and efficient procedure.

Lessons from nature--protein fibers.

Silks are protein fibers with remarkable mechanical properties. The discovery of the structural features that govern these properties is a challenge for biochemistry and structural biology. This review summarizes the results of the biochemistry of silk proteins as well as the knowledge of the molecular biology of the respective genes. In addition, an overview is presented on the efforts to produce recombinant silk proteins by biotechnological techniques.

The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action.

The serine proteinase plasmin is the key fibrinolytic enzyme that dissolves blood clots and also promotes cell migration and tissue remodeling. Here, we report the 2.65 A crystal structure of a ternary complex of microplasmin-staphylokinase bound to a second microplasmin. The staphylokinase 'cofactor' does not affect the active-site geometry of the plasmin 'enzyme', but instead modifies its subsite specificity by providing additional docking sites for enhanced presentation of the plasminogen 'substrate' to the 'enzymes's' active site. The activation loop of the plasmin 'substrate', cleaved in these crystals, can be reconstructed to show how it runs across the active site of the plasmin 'enzyme' prior to activation cleavage. This is the first experimental structure of a productive proteinase-cofactor-macromolecular substrate complex. Furthermore, it provides a template for the design of improved plasminogen activators and plasmin inhibitors with considerable therapeutical potential.

NH2-terminal structural motifs in staphylokinase required for plasminogen activation.

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.

Nuclear magnetic resonance solution structure of the plasminogen-activator protein staphylokinase.

Staphylokinase, a 15.5 kDa protein from Staphylococcus aureus, is a plasminogen activator which is currently undergoing clinical trials for the therapy of myocardial infarction and peripheral thrombosis. The three-dimensional (3D) NMR solution structure has been determined by multidimensional heteronuclear NMR spectroscopy on uniformly 15N- and 15N,13C-labeled samples of staphylokinase. Structural constraints were obtained from 82 3JHNH alpha as well as 22 3JNH beta scalar coupling constants and 2345 NOE cross-peaks, derived from 15N-edited and 13C-edited 3D NOE spectra. NOE cross-peak assignments were confirmed by analysis of ¿15N,13C¿-edited and ¿13C,13C¿-edited 4D NOE spectra. The structure is presented as a family of 20 conformers which show an average rmsd of 1.02 +/- 0.15 A from the mean structure for the backbone atoms. The tertiary structure of staphylokinase shows a well-defined global structure consisting of a central 13-residue alpha-helix flanked by a two-stranded beta-sheet, both of which are located above a five-stranded beta-sheet. Two of the connecting loops exhibit a higher conformational heterogeneity. Overall, staphylokinase shows a strong asymmetry of hydrophilic and hydrophobic surfaces. The N-terminal sequence, including Lys10 which is the site of the initial proteolytic cleavage during activation of plasminogen, folds back onto the protein core, thereby shielding amino acids with functional importance in the plasminogen activation process. From a comparison of the structure with mutational studies, a binding region for plasminogen is proposed.

NMR secondary structure of the plasminogen activator protein staphylokinase.

Staphylokinase (Sak) is a 15.5 kDa protein secreted by several strains of Staphylococcus aureus. Due to its ability to convert plasminogen, the inactive proenzyme of the fibrinolytic system, into plasmin, Sak is presently undergoing clinical trials for blood clot lysis in the treatment of thrombovascular disorders. With a view to developing a better understanding of the mode of action of Sak, we have initiated a structural investigation of Sak via multidimensional heteronuclear NMR spectroscopy employing uniformly 15N- and 15N, 13C-labelled Sak. Sequence-specific resonance assignments have been made employing 15N-edited TOCSY and NOE experiments and from HNCACB, CBCA(CO)NH, HBHA-(CBCACO) NH and CC(CO)NH sets of experiments. From an analysis of the chemical shifts, 3JHNH alpha scalar coupling constants, NOEs and HN exchange data, the secondary structural elements of Sak have been characterized.

Staphylokinase requires NH2-terminal proteolysis for plasminogen activation.

Staphylokinase (Sak), a single-chain protein comprising 136 amino acids with NH2-terminal sequence,SSSFDKGKYKKGDDA forms a complex with plasmin, that is endowed with plasminogen activating properties. Plasmin is presumed to process mature (high molecular weight, HMW) Sak to low molecular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys10-Lys11 peptide bond, but the kinetics of plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH2-terminal proteolysis of Sak for the induction of plasminogen activating potential was studied by mutagenesis of Lys10 and Lys11 in combination with NH2-terminal microsequence analysis of equimolar mixtures of Sak and plasminogen and determination of kinetic parameters of plasminogen activation by catalytic amounts of Sak. Substitution of Lys10 with Arg did not affect processing of the Arg10-Lys11 site nor plasminogen activation, whereas substitution with His resulted in cleavage of the Lys11-Gly12 peptide bond and abolished plasminogen activation. Substitution of Lys11 with Arg did not affect Lys10-Arg11 processing or plasminogen activation, whereas replacement with His did not prevent Lys10-His11 hydrolysis but abolished plasminogen activation. Substitution of Lys11 with Cys yielded an inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH2-terminal amino acids did not affect plasminogen activation, but additional deletion of Lys11 eliminated plasminogen activation. Thus generation of plasminogen activator potential in Sak proceeds via plasmin-mediated removal of the 10 NH2-terminal amino acids with exposure of Lys11 as the new NH2 terminus. This provides a structural basis for the hypothesis, derived from kinetic measurements, that plasminogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.

Functional significance of NH2- and COOH-terminal regions of staphylokinase in plasminogen activation.

Structure/function relationships in the activation of plasminogen with staphylokinase were studied using mutants of recombinant staphylokinase (Sak42D). Deletion of up to 10 NH2-terminal amino acids (Sak42D delta N10) did not affect plasminogen activation, but removal of 11 amino acids completely abolished the ability to activate plasminogen. Elimination of potential plasmin cleavage sites in the NH2-terminal region yielding mutants Sak42D(K8H,K10H,K11H) and Sak42D(K6H,K8H,K11H) did not alter the rate of the exposure of a proteolytically active site (amidolytic activity) in equimolar mixtures with plasminogen, but destroyed the plasminogen activator properties of these muteins. Deleting two residues following the preferred processing site at position 10 (Sak42 delta (K11,G12)) resulted in a mutein also inactive in plasminogen activation. Removal of the COOH-terminal Lys136, yielding Sak42D delta C1, or of Lys135 and Lys136 in Sak42D delta C2 resulted in proteins with strongly reduced plasminogen activation capacity. In contrast, substitution of Lys135 and Lys136 with Ala in Sak42D(K135A,K136A) did not affect activation. Cyanogen bromide cleavage of Sak42D(M26L,E61M,D82E) produced a 61 amino acid NH2-terminal and a 65 amino acid COOH-terminal fragment which did not activate plasminogen, but bound to plasminogen with affinity constants Ka of 4.0 x 10(5) M-1 and 1.4 x 10(7) M-1, respectively (as compared to a Ka of 1.1 x 10(8) M-1 for Sak42D). These results indicate that Lys11 and the COOH-terminal region of staphylokinase play a key role in the activation of plasminogen.

A correlation between thermal stability and structural features of staphylokinase and selected mutants: a Fourier-transform infrared study.

Variants of recombinant staphylokinase (Sak) were investigated by Fourier-transform infrared spectroscopy: Sak (wild type), Sak-M26A, Sak-M26L, and Sak-G34S/R36G/R43H (Sak-B). Estimation of the secondary structure and hydrogen-deuterium exchange experiments revealed the existence of fast-exchanging and strongly solvent-exposed fractions of the helical structures in the two samples Sak and Sak-M26L. These two samples are also thermally less stable with unfolding transition temperatures of 43.7 degrees C (Sak) and 43.5 degrees C (Sak-M26L), respectively. On contrast, Sak-M26A and Sak-G34S/R36G/R43H have a slower hydrogen-deuterium exchange, have a smaller solvent-exposed portion of the helical part, and are more resistant against thermal unfolding; the transition temperatures are 51.7 degrees C and 59.3 degrees C, respectively. The secondary structure analysis was performed by two different approaches, by curve-fitting after band narrowing and by pattern recognition (factor analysis) based upon reference spectra of proteins with known crystal structure. Within the limits of the used methods, we are unable to detect significant differences in the secondary structure of the four variants of Sak. According to the results of the factor analysis, the portions of secondary structure elements were obtained to 16-20% alpha-helix, 28-30% beta-sheet, 23-27% turns, 28-30% irregular (random) and other structure. The sharp differences in the specific plasminogen-activating capacity (Sak, Sak-G34S/R36G/R43H and Sak-M26L are fully active, but Sak-M26A does not form a stable complex with plasminogen) are not reflected in the structural features revealed by the infrared spectra of this study.

Structure-function relationships in staphylokinase as revealed by "clustered charge to alanine" mutagenesis.

Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine ("clustered charge-to-alanine scan") were characterized. Fifteen of these mutants had specific plasminogen-activating activities of > 20% of that of wild-type SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A (SakSTAR13), SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakSTAR67) had specific activities of 3%. SakSTAR13 had an intact affinity for plasminogen and a normal rate of active site exposure in equimolar mixtures with plasminogen. The plasmin-SakSTAR13 complex had a 14-fold reduced catalytic efficiency for plasminogen activation but was 5-fold more efficient for conversion of plasminogen-SakSTAR13 to plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for plasminogen and a markedly reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic efficiency toward plasminogen. Thus, plasminogen activation by catalytic amounts of SakSTAR is dependent on complex formation between plasmin(ogen) and SakSTAR, which is deficient with SakSTAR48 and SakSTAR67, but also on the induction of a functional active site configuration in the plasmin-SakSTAR complex, which is deficient with all three mutants. These findings support a mechanism for the activation of plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts plasminogen to plasmin and, more rapidly, inactive plasminogen-SakSTAR to plasmin-SakSTAR.

The fibrinolytic activity of staphylokinase mutants in the fibrin plate assay.

The fibrinolytic activity of the new plasminogen activator, recombinant staphylokinase, was compared to that of streptokinase and tissue-type plasminogen activator in the fibrin plate assay. The pattern of fibrinolysis by staphylokinase on fibrin plates differs from the other plasminogen activators. A number of mutants of staphylokinase with various amino acids in position 26 substituted for methionine in wild-type staphylokinase were compared with respect to their fibrinolytic potencies. Only the mutants with cysteine or leucine in this position have a fibrinolytic activity comparable to wild-type staphylokinase. The results on the fibrinolytic activities in the fibrin plate assay correlate with those of a plasmin generation assay, the latter is, however, less sensitive.

The thermostability of natural variants of bacterial plasminogen-activator staphylokinase.

Three natural variants (wild-type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase) of the bacterial plasminogen-activator staphylokinase, a 136-amino-acid protein secreted by certain Staphylococcus aureus strains, have been characterized. These variants differ at amino acid positions 34, 36 and 43 only, and have a very similar plasminogen-activating capacity and conformation in solution, as revealed by fluorescence spectroscopy, dynamic light scattering and circular dichroism. However, the thermostability of these variants is significantly different. At 70 degrees C and 0.5 mg protein/ml, irreversible inactivation occurred with apparent half-life (t1/2) values 0.54 +/- 0.13, 0.81 +/- 0.20 and 3.7 +/- 0.7 h (mean +/- SEM) for wild-type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase, respectively, with corresponding values at 0.08 mg/ml of 5.3 +/- 1.4 h and 11 +/- 2.0 h for wild-type staphylokinase and [R36G, R43H]staphylokinase, respectively. Dynamic light-scattering measurements indicated that inactivation was associated with protein aggregation, which precluded accurate determination of transition temperatures and enthalpies of unfolding. 0.08-0.34 mg/ml [G34S, R36G, R43H]staphylokinase, however, did not aggregate at 70 degrees C but underwent unfolding as revealed by a 20% increase in the Stokes' radius and a 30% decrease in circular dichroism. The unfolding was reversible upon cooling and was associated with full recovery of functional activity. Thus, these natural variants of staphylokinase have a different sensitivity to thermal inactivation, that is mediated by reversible unfolding of the protein and concentration-dependent irreversible aggregation. [G34S, R36G, R43H]staphylokinase, the most resistant natural variant, has a stability approaching the minimal requirements for pasteurization, which would facilitate its development for clinical use.

High yield production and purification of recombinant staphylokinase for thrombolytic therapy.

Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an ATG start codon and which express staphylokinase under the control of a tac promoter and two Shine-Dalgarno sequences in tandem. Induction of transfected E. coli TGl cells in a bacterial fermentor produced intracellular staphylokinase representing 10 to 15% of total cell protein. Gram quantities of highly purified recombinant staphylokinase were obtained from cytosol fractions by chromatography, at room temperature, on SP-Sepharose and on phenyl-Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanalization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.

Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26.

Variants of recombinant staphylokinase (Sak) were produced by site-specific mutagenesis of the unique Met-26 residue and purified to homogeneity from the cell extract of transformed E. coli. The desired mutations were confirmed by cDNA and amino-acid sequence analysis. Sak-M26L, Sak-M26C, Sak-M26R, Sak-M26V and Sak-M26A were selected for further analysis on the basis of their plasminogen activating activity. The specific fibrinolytic activities of Sak-M26L, Sak-M26C and Sak were comparable (76,000 +/- 10,000, 75,000 +/- 2400 and 78,000 +/- 9700 HU/mg, respectively; mean +/- S.E., n = 3 or 4). Active site exposure in equimolar (4.5 microM) mixtures plasminogen at room temperature was more rapid with Sak-M26L than with Sak (quantitative exposure within 4 min and 8 min, respectively). Activation of 1 microM plasminogen by catalytic amounts (5 nM) of Sak-M26L initially appeared to be somewhat faster, but comparable 50 to 60% activation was obtained within 30 min. In contrast, Sak-M26R and Sak-M26V were virtually inactive, did not form active complexes with plasminogen and did not activate plasminogen. The catalytic efficiencies for plasminogen activation were comparable for plasmin-Sak-M26L, plasmin-Sak-M26C and plasmin-Sak (0.14 microM-1 s-1, 0.16 microM-1 s-1 or 0.12 microM-1 s-1, respectively). Comparable dose-dependent lysis of 0.06 ml 125I-fibrin labeled human plasma clots submerged in 0.3 ml human plasma was obtained with Sak-M26L, Sak-M26C and Sak (concentration required for 50% lysis in 2 h, EC50, of 17 +/- 1.6 nM, 19 +/- 1.4 nM and 14 +/- 2.5 nM, respectively), whereas Sak-M26R or Sak-M26V were inactive. Sak-M26A did not form a stable complex with plasminogen, as shown by gel filtration. These data establish that substitution of the unique Met residue in position 26 of the Sak sequence with Leu or Cys has little or no influence on its plasminogen activating or fibrinolytic potential. In contrast, substitution of Met-26 with either Arg or Val results in total loss of the functional activity. Thus, the amino acid in position 26 of Sak appears to be of crucial importance for the activation of plasminogen by staphylokinase.

Mechanisms of activation of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase.

The molecular basis of the marked interspecies variability in the response of plasma fibrinolytic systems to activation by streptokinase (SK) or recombinant staphylokinase (STAR) was studied using highly purified plasminogens and alpha 2-antiplasmins from five representative species (man, baboon, rabbit, dog and cow). Human plasminogen reacted rapidly and stoichiometrically with both SK and STAR to yield potent plasminogen activators (catalytic efficiencies, kcat/Km, of 1.0 microM-1 x s-1 and 0.3 microM-1 x s-1, respectively). The complex with SK was insensitive to alpha 2-antiplasmin, which, however, rapidly inhibited the complex with STAR (second-order rate constant, k1,app of 8 x 10(6) M-1 x s-1). In a system composed of a 0.06-ml 125I-fibrin-labeled plasma clot submerged in 0.30 ml plasma, both SK and STAR had potent fibrinolytic properties, causing 50% clot lysis in 2 h (EC50), with 120 nM and 13 nM, respectively. Clot lysis with SK was non-fibrin specific (residual fibrinogen < 10%), whereas lysis with STAR was highly fibrin specific (residual fibrinogen 76%). Canine plasminogen reacted avidly with SK, but SK was rapidly degraded; it reacted rapidly and quantitatively with STAR to form a potent plasminogen-activating complex (kcat/Km of 0.4 microM-1 x s-1) which was sensitive to neutralization by alpha 2-antiplasmin (k1,app of 6 x 10(5) M-1 x s-1). In a canine plasma milieu, SK was relatively potent (EC50 200 nM) and fibrin specific, whereas STAR was very potent (EC50 1.3 nM) but poorly fibrin specific. Baboon and rabbit plasminogen did not form stable stoichiometric complexes with SK, but reacted stoichiometrically and quantitatively with STAR. The complexes with STAR, however, had low catalytic efficiencies for the activation of their autologous plasminogens (kcat/Km 0.02 microM-1 x s-1) and reacted more slowly with alpha 2-antiplasmin (k1,app 5-10 x 10(5) M-1 x s-1). Bovine plasminogen was virtually unreactive towards both SK and STAR as well as to their complexes with human plasminogen, as monitored by measurement of the initial activation rates. The resistance to fibrinogen degradation with STAR observed in the human system could be transferred to the canine system by reconstituting canine plasma, depleted of plasminogen and alpha 2-antiplasmin, with the human proteins. Conversely, the sensitivity to fibrinogen degradation of the canine system could be transferred to the human system by reconstituting depleted plasma with canine plasminogen and alpha 2-antiplasmin. It is concluded that the variability in the response of mammalian plasma fibrinolytic systems to activation with SK or STAR is determined mainly by the extent of complex formation of these compounds with plasminogen, by the catalytic efficiencies of the complexes for the activation of autologous plasminogen and by the rate of inhibition of these complexes by alpha 2-antiplasmin.

Physical and conformational properties of staphylokinase in solution.

The structure of staphylokinase has been analyzed by solution X-ray scattering, dynamic light scattering, ultracentrifugation and ultraviolet circular dichroism spectroscopy. Staphylokinase has a radius of gyration of 2.3 nm, a Stokes radius of 2.12 nm and a maximum dimension of 10 nm. The sedimentation coefficient is 1.71 S. These physical parameters indicate that the shape of staphylokinase is very elongated. The protein molecule consists of two folded domains of similar size. The mean distance of the centres of gravity of the domains is 3.7 nm. The mutual positions of the two domains are variable in solution. Thus, the molecule is shaped like a flexible dumbbell. About 18% of the amino acids of staphylokinase are organized in helical structures, 30% are incorporated in beta-sheets and 20% form turns.