Pro42 and Val45 of staphylokinase modulate intermolecular interactions of His43-Tyr44 pair and specificity of staphylokinase-plasmin activator complex

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with plasmin (Pm) and changes its substrate specificity to create a plasminogen (Pg) activator complex. The His(43)-Tyr(44) pair of SAK resides within the active site cleft of the partner Pm and generates intermolecular contacts to confer Pg activator ability to the SAK-Pm bimolecular complex. Site-directed mutagenesis and molecular modeling studies unravelled that mutation at 42nd or 45th positions of SAK specifically disrupts cation-pi interaction of His(43) with Trp(215) of partner Pm within the active site, whereas pi-pi interaction of Tyr(44) with Trp(215) remain energetically favoured.     

Role of the N-terminal region of staphylokinase (SAK): evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complex formation

Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), which requires both the conversion of PG to plasmin (Pm) to expose an active site in PG-SAK activator complex and the amino-terminal processing of SAK to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of SAK affects its PG activation capability was investigated by generating SAK mutants, blocked in the native amino-terminal processing site of SAK, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilized PG-SAK binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with PG, Pm or miniplasmin substrate during ternary complex formation as compared to native SAK. Additionally, activator complex formed with PRM3 failed to activate miniplasminogen and exhibited highly diminished activation of substrate PG. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of SAK in accession and processing of substrate by the SAK-Pm activator complex to potentiate the PG activation by enhancing and/or stabilizing the interaction of free PG.     

Enhanced plasminogen activation by staphylokinase in the presence of streptokinase beta/betagamma domains: plasminogen kringles play a role

Presence of isolated beta or betagamma domains of streptokinase (SK) increased the catalytic activity of staphylokinase (SAK)-plasmin (Pm) complex up to 60%. In contrast, fusion of SK beta or betagamma domains with the C-terminal end of SAK drastically reduced the catalytic activity of the activator complex. The enhancement effect mediated by beta or betagamma domain on Pg activator activity of SAK-Pm complex was reduced greatly (45%) in the presence of isolated kringles of Pg, whereas, kringles did not change cofactor activity of SAK fusion proteins (carrying beta or betagamma domains) significantly. When catalytic activity of SAK-microPm (catalytic domain of Pm lacking kringle domains) complex was examined in the presence of isolated beta and betagamma domains, no enhancement effect on Pg activation was observed, whereas, enzyme complex formed between microplasmin and SAK fusion proteins (SAKbeta and SAKbetagamma) displayed 50-70% reduction in their catalytic activity. The present study, thus, suggests that the exogenously present beta and betagamma interact with Pg/Pm via kringle domains and elevate catalytic activity of SAK-Pm activator complex resulting in enhanced substrate Pg activation. Fusion of beta or betagamma domains with SAK might alter these intermolecular interactions resulting in attenuated functional activity of SAK.     

Fibrin‐targeted plasminogen activation by plasminogen activator,PadA, from Streptococcus dysgalactiae

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three‐domain streptokinase (SK) and two‐domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2‐antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single‐domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single‐domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single‐domain plasminogen activator that activates bovine plasminogen in a fibrin‐targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin‐specific plasminogen activators for thrombolytic therapy.     

Staphylokinase as a Plasminogen Activator Component in Recombinant Fusion Proteins

The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.     

Degradation of streptokinase and the catalytic properties of the plasmin-streptokinase complex

Streptokinase (SK) interacts with human plasminogen (Pg) or plasmin (Pm) with formation of Pg-SK or Pm-SK complex. Pm-SK complex manifests a fibrinolytic, amidolytic and Pg activator activity. SK in complex with Pm isn't stable and so capable to be hydrolysed rapidly. We investigated a correlation between molecular form of SK and catalytic properties of equimolar Pm-SK complex during preincubation at 20 degrees C. It was found out that amidolytic activity of Pm-SK complex was not changing for 5 hours and decreased to the initial Pm value after 24 hours. During this time alpha 2-antiplasmin (alpha 2-AP) has any effect on amidolytic activity of the complex. Fibrinolytic activity of Pm-SK complex makes up 20% of the initial Pm value and wasn't changing within the investigated period. Pg activator activity was decreasing rapidly to 30-40% of the initial one within few minutes from the moment of Pm-SK complex formation. It was 10-20% of that initial after 24 hours. The decrease in Pg activator activity of Pm-SK complex correlated with the initial very rapid conversion of 47 kDa SK to 36 kDa SK within few minutes and following more slow conversion of SK in 31, 25 and 15 kDa fragments after 5 hours. alpha 2-AP didn't influence on the Pg activator activity of Pm-SK complex but eliminated its fibrinolytic activity completely. It was supposed that alpha 2-AP inhibited fibrinolytic activity of Pm-SK complex similarly to 6-aminohexanoic acid by preventing Pm-SK complex binding to fibrin polymer.     

Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation

Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys(414) binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK.Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys(414) deletion mutant (SKDeltaK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKDeltaK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKDeltaK414 was approximately 10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK.Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys(414) binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes.     

Novel self-cleavage activity of Staphylokinase fusion proteins: An interesting finding and its possible applications

Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli expression vectors. This finding was further investigated by cloning and expressing different SAK fusions, both native and N-terminal deletions, with fusion tags like glutathione S-transferase (GST) and signal sequence of SAK in bacterial system. While all the N-terminal SAK fusions were found to self-cleave in crude and purified preparations, the C-terminal SAK fusion was stable. The cleavage property of Staphylokinase fusion proteins, inhibited by reduced glutathione and PMSF, was independent of its thrombolytic activity and also independent on the type of host employed for its expression. The serine protease domain of the SAK gene possibly lies between 20th to 77th amino acid and serine 41 of this region appears critical for such a cleavage property.     

Crystal structure of a staphylokinase: variant a model for reduced antigenicity.

Staphylokinase (SAK) is a 15.5-kDa protein from Staphylococcus aureus that activates plasminogen by forming a 1 : 1 complex with plasmin. Recombinant SAK has been shown in clinical trials to induce fibrin-specific clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies have demonstrated the feasibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallographic evidence that the SAK(S41G) mutant may assume a dimeric structure. This dimer model, at 2.3-A resolution, could explain a major antigenic epitope (residues A72-F76 and residues K135-K136) located in the vicinity of the dimer interface as identified by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce the antigenicity of SAK.     

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.     

Modifying effect of antiplasminogen monoclonal antibody IV-1c on human plasmin catalytic properties

Antiplasminogen monoclonal antibody IV-1c (IV-1c) binds to Val 709-Gly 718 site of plasminogen (Pg) protease domain, which is far removed from the active site. Pg-IV-1c complex formation induces catalytic activity in proenzymes active site. Influence of IV-1c binding to plasmin (Pm) on Pm catalytic properties has not been investigated yet. Data on catalytic properties of Pm in equimolar Pm-IV-1c complex are presented. It was found that Pm and mini-Pm amidolytic and caseinolytic activity was twice as high as in Pm-IV-1c and mini-Pm-IV-1c complexes. 20 mM 6-AHA and 100 mM arginine did not influence this rise. The increase of amidolytic activity is connected with reduction of K(m) of S 2251 hydrolysis reaction for Pm and mini-Pm from 0.125 and 0.43 to 0.05 and 0.23 mM, correspondingly. Kcat remains almost the same. Fibrinolytic and fibrinogenolytic activity of Pm in Pm-IV-1c complex decreased to 20% of initial value alpha 2-Antiplasmin inhibited Pm activity in complex Pm-IV-1c by 80%. Pm-IV-1c complex did not activate free Pg, but activated equimolar Pg-IV-1c complex. Affinity of IV-1c to Pm and Pg was the same as C50 approximately 1.5 nM. Binding of Pm with IV-1c in a complex: a) leads to increase of Pm active site affinity to LMW substrates; b) causes steric hindrances for fibrin/fibrinogen access to Pm active site; c) proceeds with the same affinity for Pm and Pg, that indicates to invariable Val 709-Gly 718 site conformation after Pg transition in Pm.     

Computational simulations assessment of mutations impact on streptokinase (SK) from a group G streptococci with enhanced activity – insights into the functional roles of structural dynamics flexibility of SK and stabilization of SK–μplasmin catalytic complex

Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasmin (Pm), is a protein secreted by groups A, C, and G streptococci (GAS, GCS, and GGS, respectively), with high sequence divergence and functional heterogeneity. While roles of some residual changes in altered SK functionality are shown, the underlying structural mechanisms are less known. Herein, using computational approaches, we analyzed the conformational basis for the increased activity of SK from a GGS (SKG132) isolate with four natural residual substitutions (Ile33Phe, Arg45Gln, Asn228Lys, Phe287Ile) compared to the standard GCS (SKC). Using the crystal structure of SK.Pm catalytic complex as main template SKC.μPm catalytic complex was modeled through homology modeling process and validated by several online validation servers. Subsequently, SKG132.μPm structure was constructed by altering the corresponding residual substitutions. Results of three independent MD simulations showed increased RMSF values for SKG132.μPm, indicating the enhanced structural flexibility compared to SKC.μPm, specially in 170 and 250 loops and three regions: R1 (149–161), R2 (182–215) and R3 (224–229). In parallel, the average number of Hydrogen bonds in 170 loop, R2 and R3 (especially for Asn228Lys) of SKG132 compared to that of the SKC was decreased. Accordingly, residue interaction networks (RINs) analyses indicated that Asn228Lys might induce more level of structural flexibility by generation of free Lys256, while Phe287Ile and Ile33Phe enhanced the stabilization of the SKG132.μPm catalytic complex. These results denoted the potential role of the optimal dynamic state and stabilized catalytic complex for increased PA potencies of SK as a thrombolytic drug.     

Powdery mildew resistance gene <Emphasis Type="Italic">Pm22</Emphasis> in cultivar Virest is a member of the complex <Emphasis Type="Italic">Pm1</Emphasis> locus in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L. em Thell.)

The powdery mildew resistance gene Pm22, identified in the Italian wheat cultivar Virest and originally assigned to wheat chromosome 1D, was mapped to chromosome 7A with the aid of molecular markers. Mapping of common AFLP and SSR markers in two wheat crosses segregating for Pm22 and Pm1c, respectively, indicated that Pm22 is a member of the complex Pm1 locus. Pm22 also showed a pattern of resistance reaction to a differential set of Blumeria graminis f. sp. tritici isolates that was distinguishable from those from other Pm1 alleles in lines Axminster/8*Cc ( Pm1a), MocZlatka ( Pm1b), Weihenstephan Stamm M1N ( Pm1c) and Triticum spelta var. duhamelianum TRI 2258 ( Pm1d). Based on these results, the gene symbol Pm1e is proposed for the powdery mildew resistance gene in cv. Virest.     

Enhancement of secretion and extracellular stability of staphylokinase in Bacillus subtilis by wprA gene disruption

Staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.     

Mechanism of fibrin-specific fibrinolysis by staphylokinase: participation of alpha 2-plasmin inhibitor

When the extent of plasminogen activation by staphylokinase (SAK) or streptokinase (SK) was measured in human plasma, SAK barely induced plasminogen activation, whereas SK activated plasminogen significantly. When the plasma was clotted with thrombin, the plasminogen activation by SAK was markedly enhanced, but that of SK was little enhanced. Similarly, in a purified system composed of plasminogen, fibrinogen and alpha 2-plasmin inhibitor (alpha 2-PI, alpha 2-antiplasmin), such a fibrin clot increased the activity of SAK significantly. However, when alpha 2-PI was removed from the reaction system, enhancement of the SAK reaction was not observed. In addition, SAK as distinct from SK, showed very little interference with the action of alpha 2-PI. Plasminogen activation by SAK is thus essentially inhibited by alpha 2-PI, but this reaction is not inhibited in fibrin clots. These results suggest that SAK forms a complex with plasminogen, which binds to fibrin and induces fibrinolysis.     

Isolation of cDNA clones corresponding to genes differentially expressed in pericarp of mume (Prunus mume) in response to ripening, ethylene and wounding signals

Ripening of climacteric fruit is a complex developmental process that includes many changes in gene expression. Some ripening-regulated genes are responsive to ethylene and/or wounding signals. Wounding increased Pm-ACS1 expression in Prunus mume (Japanese apricot), but was negatively regulated by ethylene. However, exposure of freshly harvested mature green mume fruit to ethylene induced PmACS1 . Fifteen complementary DNA clones corresponding to messenger RNAs differentially expressed in the pericarp of P. mume fruit in response to ripening, ethylene and wounding signals were isolated by differential display. Quantitative real-time PCR analysis distinctly showed that these genes are differentially regulated. Genes that were upregulated during fruit ripening include Pm15 (cinnamyl-alcohol dehydrogenase), Pm21 (2-oxoacid-dependent dioxygenase), Pm22 (1-acyl- sn -glycerol-3-phosphate acyltransferase), Pm27 (unknown function), Pm38 (alcohol dehydrogenase), Pm41 (no homology), Pm52 (no homology), Pm65 (pectate lyase), Pm68 (expansin), Pm69 (serine carboxypeptidase) and Pm94 (alcohol acyltransferase). Expression of most of these genes was also inducible by ethylene and some of them were inducible by wounding. Pm3 (water channel protein, MIP) and Pm8 (unknown function) were downregulated during ripening. Expression of Pm71 (no homology) and Pm74 (NAC family protein) did not increase during ripening or in response to ethylene, but was upregulated in response to wounding. The possible physiological roles of these genes during ripening and in response to ethylene and wounding are discussed.     

Enhancement of Secretion and Extracellular Stability of Staphylokinase in Bacillus subtilis by wprA Gene Disruption

Staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.     

PmST2: a novel Pasteurella multocida glycolipid α2-3-sialyltransferase

Pasteurella multocida (Pm) is a multi-species pathogen that causes diseases in animals and humans. Sialyltransferase activity has been detected in multiple Pm strains and sialylation has been shown to be important for the pathogenesis of Pm. Three putative sialyltransferase genes have been identified in Pm genomic strain Pm70. We have reported previously that a Pm0188 gene homolog in Pm strain P-1059 (ATCC 15742) encodes a multifunctional sialyltransferase (PmST1). We demonstrate here that while PmST1 prefers to use oligosaccharides as acceptors, PmST2 encoded by the Pm0508 gene homolog in the same Pm strain is a novel glycolipid α2-3-sialyltransferase that prefers to use lactosyl lipids as acceptor substrates. PmST2 and PmST1 thus complement each other for an efficient synthesis of α2-3-linked sialosides with or without lipid portion. In addition, β1-4-linked galactosyl lipids are better PmST2 substrates than β1-3-linked galactosyl lipids. PmST2 has been used successfully in the preparative scale synthesis of sialyllactosyl sphingosine (lyso-GM3), which is an important glycolipid and an intermediate for synthesizing more complex glycolipids such as gangliosides.     

具有抗血小板聚集功能的新型葡激酶突变体的表达、纯化及性质

[目的]为解决溶栓后再栓塞问题,构建N-端含RGD(Arg-Gly-Asp)序列的葡激酶双功能突变体.研究突变体的表达和纯化,并进行性质分析.[方法]将突变后的葡激酶突变体序列连入pBV220质粒,转化大肠杆菌BL21进行表达.阳离子交换、凝胶过滤和阴离子交换三步层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定,并测定纯化产物对血小板聚集的抑制效应.[结果]PAGE扫描结果显示,葡激酶突变体蛋白在大肠杆菌BL21中的表达量约占菌体蛋白总量的40%～50%;三步层析纯化后,HPLC测定其纯度可达95%.酪蛋白凝胶板溶圈法测得其比活性分别为10.8×104和11.0×104HU/mg,与野生型葡激酶活性相当;且具有明显的抗血小板聚集活性,血小板聚集仪测定其血小板聚集抑制率分别为10.72%和19.71%,明显高于野生型葡激酶血小板聚集抑制率.本实验利用pBV220载体高效表达了葡激酶突变体基因,得到了高纯度、高活性的突变体蛋白,为葡激酶生产产业化和临床应用奠定了良好的基础.