A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m210919200
ISSN1083-351X
AutoresSau‐Ching Wu, Francis Castellino, Sui‐Lam Wong,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoTo develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein inBacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced fromB. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues. To develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein inBacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced fromB. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues. tissue plasminogen activator ε-amino-N-caproic acid platelet poor plasma staphylokinase staphylokinase-linker-Kringle-1 fusion time required for 50% clot lysis matrix-assisted laser desorption ionization-time of flight mass spectrometry enzyme-linked immunosorbent assay HEPES-buffered saline Thrombolysis (1Guzman L.A. Lincoff A.M. J. Thromb. Thrombolysis. 1997; 4: 337-343Crossref PubMed Scopus (1) Google Scholar, 2Hennekens C.H. O'Donnell C.J. Ridker P.M. Marder V.J. J. Am. Coll. Cardiol. 1995; 25 Suppl. 7: S18-S22Crossref Scopus (32) Google Scholar, 3Tsikouris J.P. Tsikouris A.P. Pharmacotherapy. 2001; 21: 207-217Crossref PubMed Scopus (37) Google Scholar, 4Sinnaeve P. van de W.F. Thromb. Res. 2001; 103 Suppl. 1: S71-S79Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) is one of the well established treatments for patients with acute myocardial infarction (commonly known as heart attack). Blood clot-dissolving agents currently approved for thrombolytic therapy include tissue plasminogen activator (tPA),1 urokinase, streptokinase, and their derivatives (3Tsikouris J.P. Tsikouris A.P. Pharmacotherapy. 2001; 21: 207-217Crossref PubMed Scopus (37) Google Scholar, 4Sinnaeve P. van de W.F. Thromb. Res. 2001; 103 Suppl. 1: S71-S79Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Although the treatment can reduce mortality, several large scale clinical trials indicate that these blood clot-dissolving agents are far from ideal (4Sinnaeve P. van de W.F. Thromb. Res. 2001; 103 Suppl. 1: S71-S79Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 5The GUSTO Angiographic Investigators N. Engl. J. Med. 1993; 329: 1615-1622Crossref PubMed Scopus (1884) Google Scholar). Even with tPAs, only about 60% of the treated patients have their blood flow restored 90 min after the onset of treatment (5The GUSTO Angiographic Investigators N. Engl. J. Med. 1993; 329: 1615-1622Crossref PubMed Scopus (1884) Google Scholar). Some of the treated patients will suffer from reocclusion or bleeding complications such as hemorrhagic strokes (2Hennekens C.H. O'Donnell C.J. Ridker P.M. Marder V.J. J. Am. Coll. Cardiol. 1995; 25 Suppl. 7: S18-S22Crossref Scopus (32) Google Scholar, 3Tsikouris J.P. Tsikouris A.P. Pharmacotherapy. 2001; 21: 207-217Crossref PubMed Scopus (37) Google Scholar, 6The InTIME-II Investigators Eur. Heart J. 2000; 21: 2005-2013Crossref PubMed Scopus (182) Google Scholar). Furthermore, the reoccluded clots are usually platelet-rich and are more resistant to tPA-mediated clot lysis (7Zhu Y. Carmeliet P. Fay W.P. Circulation. 1999; 99: 3050-3055Crossref PubMed Scopus (159) Google Scholar, 8Serizawa K. Urano T. Kozima Y. Takada Y. Takada A. Thromb. Res. 1993; 71: 289-300Abstract Full Text PDF PubMed Scopus (17) Google Scholar). Hence, choices of more potent blood clot dissolving agents, which provide a rapid, complete, and sustained reperfusion with minimal side effects, are needed. Staphylokinase (SAK), a 136-amino acid protein from certain lysogenicStaphylococcus aureus strains, is a plasminogen activator and a promising blood clot-dissolving agent with clinical potency that is at least as good as tPA (9Vanderschueren S. Barrios L. Kerdsinchai P. Van den Heuvel P. Hermans L. Vrolix M. De Man F. Benit E. Muyldermans L. Collen D. Van de Werf F. Circulation. 1995; 92: 2044-2049Crossref PubMed Scopus (163) Google Scholar, 10Vanderschueren S. Dens J. Kerdsinchai P. Desmet W. Vrolix M. De Man F. Van den Heuvel P. Hermans L. Collen D. Van de Werf F. Am. Heart J. 1997; 134: 213-219Crossref PubMed Scopus (65) Google Scholar). In addition, it has some desirable features that are superior to tPA (11Collen D. Nat. Med. 1998; 4: 279-284Crossref PubMed Scopus (153) Google Scholar). Notably, SAK mediates the lysis of platelet-rich and retracted clots efficiently (12Suehiro A. Tsujioka H. Yoshimoto H. Ueda M. Higasa S. Kakishita E. Thromb. Res. 1995; 80: 135-142Abstract Full Text PDF PubMed Scopus (5) Google Scholar, 13Hauptmann J. Glusa E. Blood Coagul. Fibrinolysis. 1995; 6: 579-583Crossref PubMed Scopus (13) Google Scholar) and shows exceptional fibrin specificity (9Vanderschueren S. Barrios L. Kerdsinchai P. Van den Heuvel P. Hermans L. Vrolix M. De Man F. Benit E. Muyldermans L. Collen D. Van de Werf F. Circulation. 1995; 92: 2044-2049Crossref PubMed Scopus (163) Google Scholar, 10Vanderschueren S. Dens J. Kerdsinchai P. Desmet W. Vrolix M. De Man F. Van den Heuvel P. Hermans L. Collen D. Van de Werf F. Am. Heart J. 1997; 134: 213-219Crossref PubMed Scopus (65) Google Scholar, 14Sakharov D.V. Lijnen H.R. Rijken D.C. J. Biol. Chem. 1996; 271: 27912-27918Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 15Silence K. Collen D. Lijnen H.R. J. Biol. Chem. 1993; 268: 9811-9816Abstract Full Text PDF PubMed Google Scholar). These properties can help minimize reocclusion and bleeding complications. Much has been studied on the action of SAK in vivo. To function as the plasminogen activator, SAK first forms a complex with plasmin(ogen) (16Parry M.A. Fernandez-Catalan C. Bergner A. Huber R. Hopfner K.P. Schlott B. Guhrs K.H. Bode W. Nat. Struct. Biol. 1998; 5: 917-923Crossref PubMed Scopus (134) Google Scholar). Complex formation is followed by SAK processing in which the N-terminal peptide containing the first 10 amino acids from SAK is removed by cleaving at a twin lysine site between residues 10 and 11 (Fig. 1A). This processing step is essential for attaining an active form of SAK (17Gase A. Hartmann M. Guhrs K.H. Rocker A. Collen D. Behnke D. Schlott B. Thromb. Haemostasis. 1996; 76: 755-760Crossref PubMed Scopus (18) Google Scholar, 18Schlott B. Guhrs K.H. Hartmann M. Rocker A. Collen D. J. Biol. Chem. 1998; 273: 22346-22350Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19Szarka S. Sihota E. Habibi H.R. Wong S.-L. Appl. Environ. Microbiol. 1999; 65: 506-513Crossref PubMed Google Scholar). The SAK-plasminogen complex then forms a ternary complex with another molecule of plasminogen and converts this plasminogen to plasmin. When the SAK-plasmin complex is not fibrin-bound, it can be inhibited by the natural plasmin inhibitor, α2-antiplasmin, present in plasma. In contrast, the fibrin-bound plasminogen activator complex is much more resistant to α2-antiplasmin-mediated inhibition (15Silence K. Collen D. Lijnen H.R. J. Biol. Chem. 1993; 268: 9811-9816Abstract Full Text PDF PubMed Google Scholar). The result is a preferential plasminogen activation by SAK at the fibrin surface that contributes to the fibrin specificity of SAK in a plasma milieu. This fibrin specificity is made even stronger by the preferential binding of SAK to plasmin(ogen) that is fibrin-bound (14Sakharov D.V. Lijnen H.R. Rijken D.C. J. Biol. Chem. 1996; 271: 27912-27918Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The fibrin-specific property of SAK underlies an interesting observation in clinical trials. The fibrinogen levels in patients treated with SAK remain close to 100%, whereas patients treated with tPA have 32% of fibrinogen cleaved and degraded in their plasma (9Vanderschueren S. Barrios L. Kerdsinchai P. Van den Heuvel P. Hermans L. Vrolix M. De Man F. Benit E. Muyldermans L. Collen D. Van de Werf F. Circulation. 1995; 92: 2044-2049Crossref PubMed Scopus (163) Google Scholar, 10Vanderschueren S. Dens J. Kerdsinchai P. Desmet W. Vrolix M. De Man F. Van den Heuvel P. Hermans L. Collen D. Van de Werf F. Am. Heart J. 1997; 134: 213-219Crossref PubMed Scopus (65) Google Scholar). Although SAK is a fibrin-specific thrombolytic agent, it has no fibrin binding ability by itself. It binds to fibrin clots only indirectly through the interaction with any clot-bound plasmin(ogen). It would be of interest to determine whether the clot lysis efficacy of SAK can be improved by engineering it with direct fibrin binding ability. Faster clot lysis will restore blood flow in a more timely manner and reduce damage to heart tissues. Furthermore, if less SAK is required to achieve the same degree of clot lysis, the risk for side effects can be minimized. Plasminogen binds to fibrin clots via its Kringle domains at the N-terminal regions (20Castellino F.J. McCance S.G. CIBA Found. Symp. 1997; 212: 46-60PubMed Google Scholar). These Kringle domains are ∼80 amino acids long; some of them possess the lysine binding capability (21Hoover G.J. Menhart N. Martin A. Warder S. Castellino F.J. Biochemistry. 1993; 32: 10936-10943Crossref PubMed Scopus (37) Google Scholar, 22Wu T.P. Padmanabhan K.P. Tulinsky A. Blood Coagul. Fibrinolysis. 1994; 5: 157-166Crossref PubMed Scopus (42) Google Scholar, 23Mathews I.I. Vanderhoff-Hanaver P. Castellino F.J. Tulinsky A. Biochemistry. 1996; 35: 2567-2576Crossref PubMed Scopus (73) Google Scholar). Thus, plasminogen binds abundantly to fibrin clots during clot lysis since more C-terminal lysine residues are generated through the action of plasmin, a trypsin-like protease that cuts after lysine and arginine residues. Among the characterized Kringles from human plasminogen (24Lerch P.G. Rickli E.E. Lergier W. Gillessen D. Eur. J. Biochem. 1980; 107: 7-13Crossref PubMed Scopus (183) Google Scholar, 25Menhart N. Sehl L.C. Kelley R.F. Castellino F.J. Biochemistry. 1991; 30: 1948-1957Crossref PubMed Scopus (84) Google Scholar, 26Sehl L.C. Castellino F.J. J. Biol. Chem. 1990; 265: 5482-5486Abstract Full Text PDF PubMed Google Scholar, 27Chang Y. Mochalkin I. McCance S.G. Cheng B. Tulinsky A. Castellino F.J. Biochemistry. 1998; 37: 3258-3271Crossref PubMed Scopus (89) Google Scholar, 28Marti D. Schaller J. Ochensberger B. Rickli E.E. Eur. J. Biochem. 1994; 219: 455-462Crossref PubMed Scopus (54) Google Scholar) and tPA (29Byeon I.-J.L. Kelley R.F. Mulkerrin M.G. An S.S.A. Llinás M. Biochemistry. 1995; 34: 2739-2750Crossref PubMed Scopus (21) Google Scholar), Kringle-1 from human plasminogen has the highest lysine binding affinity. Therefore, Kringle-1 was selected as the fibrin targeting domain and fused to the C-terminal end of SAK (Fig.1). To ensure that each domain within the fusion can fold independently and that each has sufficient space to interact with its target, a 20-amino acid linker is inserted between these domains. The resulting fusion (designated SAK-L-K1) and its three site-specific muteins (SAKM1-L-K1, SAKM2-L-K1, and SAKM3-L-K1) were produced via secretion from Bacillus subtilis and Pichia pastoris. Biochemical properties, clot targeting ability, and clot lysis activity of the purified SAK-L-K1 and its derivatives were determined. In comparison with SAK, the engineered SAKM3-L-K1 consistently mediated faster lysis of both fibrin and plasma clots in vitro. pSAKLK1 is a pUB18-based B. subtilisplasmid (30Wong S.-L. Gene. 1989; 83: 215-223Crossref PubMed Scopus (26) Google Scholar) for secretory production of SAK-L-K1 under control of theB. subtilis P43 promoter for transcription and the levansucrase signal sequence (SacB SP) for secretion. To introduce the linker sequence between SAK and the Kringle-1 domain from human plasminogen, the nucleotide sequence of the expression cassette (P43-SacBSP-SAK-L-K1) was split into two portions. The first portion covered from the P43 promoter to the middle half of the linker sequence. The sequence was generated by PCR using pSAKP (31Ye R. Kim J.H. Kim B.G. Szarka S. Sihota S. Wong S.-L. Biotechnol. Bioeng. 1999; 62: 87-96Crossref PubMed Google Scholar) as template. The primers used for this amplification were P43F (5′-GGGAATTCGAGCTCAGCATTATTG-3′) and SAKLB (5′-CTTGTCGACCCACCAGAAGTACTTCCTTTCTTTTCTATAACAACCTTTG-3′). The resulting product was an 895-bp EcoRI-SalI fragment. After digestion with both EcoRI andSalI, the fragment was inserted into pUB18 (30Wong S.-L. Gene. 1989; 83: 215-223Crossref PubMed Scopus (26) Google Scholar) at the corresponding sites located in the polylinker region of the vector to generate pUB18-SAKLF. The second half of the expression cassette carried a sequence encoding the C-terminal half of the linker and the Kringle-1 domain. This sequence was generated by PCR using a cDNA clone of human plasminogen as template. This cDNA clone was kindly provided by Dr. Ross T. A. MacGillivray at the Department of Biochemistry and Molecular Biology, University of British Columbia. The forward primer LK1F (5′-GGGTCGACAAGTGGTGGATCTACTAGTGGCTCTGGATCCGGAATTTGCAAGACTGGGAATGG-3′) encoded the sequence for both the C-terminal half of the linker and the beginning of the Kringle-1 domain. The backward primer LK1B (5′-CTTCTAGATTATGATCAACACTCAAGAATGTCGCAGTAG-3′) introduced a translation termination codon (TGA) at the end of the Kringle-1 sequence and an XbaI site at the 3′ end of the fragment. The resulting 310-bp SalI-XbaI PCR product was digested with SalI and XbaI and inserted into the corresponding sites in the polylinker region of the pUB18-SAKLF to generate pSAKLK1. To eliminate the N-linked glycosylation site in SAK-L-K1, Asn-28 was changed to Asp and Ala to generate SAKM1-L-K1 and SAKM2-L-K1, respectively. The change of Thr-30 to Ala resulted in the generation of SAKM3-L-K1. These mutations were generated by inverse PCR (32Hemsley A. Arnheim N. Toney M.D. Cortopassi G. Galas D.J. Nucleic Acids Res. 1989; 17: 6545-6551Crossref PubMed Scopus (416) Google Scholar) using the primer pairs shown in Table I with pSAKLK1 as the template. The entire plasmid was amplified, and the PCR product was treated with T4 polynucleotide kinase, self-ligated, and transformed toB. subtilis WB600 (33Wu X.-C. Lee W. Tran L. Wong S.-L. J. Bacteriol. 1991; 173: 4952-4958Crossref PubMed Scopus (281) Google Scholar). The resulting clones were screened for the appropriate mutation by direct sequencing of the expression cassette (P43-SacBSP-SAK-L-K1). Each of the mutated cassettes was then inserted as an EcoRI-XbaI fragment into pUB18 to generate pSAKM1LK1, pSAKM2LK1, and pSAKM3LK1. These vectors allowed the rapid examination of the effects of mutation on SAK activity in B. subtilis.Table IGlycosylation-site mutants of SAK-L-K1SAK-L-K1Glycosylation sitePrimer sequenceWild typeAsn-28-Val-Thr-30SAKM1-L-K1Asp-28-Val-Thr-30Forward primer: SAKN28F, 5′-GTTATTTTGAACCAACAGGCCCGTATTTGATGGTAG (A/C)TGTGACTGGAGT-3′Backward primer: SAKN28B, 5′-GGGCCTGTTGGTTCAAAATAAC-3′SAKM2-L-K1Ala-28-Val-Thr-30Forward primer: SAKN28FBackward primer: SAKN28BSAKM3-L-K1Asn-28-Val-Ala-30Forward primer: SAKTHRALAF, 5′-GTATTTGATGGTAAATGTGGCTGGAGTTGATGG-3′Backward primer: SAKN28BThe amino acids in bold letters designate the mutation in the glycosylation site. The degenerate mutagenic primer SAKN28F leads to the generation of mutants with Asn-28 changed to Asp or Ala. Specific nucleotides in mutagenic primers that lead to mutations are shown in bold. Open table in a new tab The amino acids in bold letters designate the mutation in the glycosylation site. The degenerate mutagenic primer SAKN28F leads to the generation of mutants with Asn-28 changed to Asp or Ala. Specific nucleotides in mutagenic primers that lead to mutations are shown in bold. Two primers (AFACSAKF and SAKK1CPICB) were designed to facilitate cloning of these structural genes immediately downstream of the α-factor signal sequence in the P. pastoris expression vector pPICZαA (Invitrogen). pSAKLK1 was used as the template for PCR amplification with AFACSAKF (5′-GACTCGAGAAGAGATCGAGCTCATTCGACAAAGG-3′) as the forward primer and SAKK1CPICB (5′-GAGCGGCCGCTTAACACTCAAGAATGTCGGAGTAG-3′) as the backward primer. A 730-bp PCR product was generated withXhoI at the 5′ end and NotI at the 3′ end. The amplified sequence was digested with XhoI andNotI and ligated to the similarly digested pPICZαA to generate pPICSAKLK1. pPICSAKLK1 was transformed to E. coliTOP10F′ and selected for resistance to zeocin (25 μg/ml) (Invitrogen). pPICSAKM2LK1 and pPICSAKM3LK1 were also generated using the same approach with pSAKM2LK1 and pSAKM3LK1 as the PCR template, respectively. pPICSAKM2LK1 or pPICSAKM3LK1 was linearized with PmeI and transformed toP. pastoris X-33 (a wild-type strain, Invitrogen) using EasyComp method according to the manufacturer's protocol. Transformants were selected on zeocin (600 and 1000 μg/ml) and screened for secretory production of SAKM2-L-K1 or SAKM3-L-K1. X-33 cells with pPICZαA and pPICSAKLK1 integrated were similarly prepared to serve as the negative and wild-type controls, respectively. For protein purification, a Pichia transformant showing the highest expression level of SAKM3-L-K1 was cultured for 16–18 h at 28–30 °C in buffered glycerol-complex medium (34Rosenfeld S.A. Methods Enzymol. 1999; 306: 154-169Crossref PubMed Scopus (43) Google Scholar). Cells were pelleted at 3,000 × g for 5 min and resuspended to 100 Klett units using a Klett-Summerson photoelectric colorimeter (Klett Mfg. Co.) in buffered methanol-complex medium (34Rosenfeld S.A. Methods Enzymol. 1999; 306: 154-169Crossref PubMed Scopus (43) Google Scholar). Growth continued at 28–30 °C in a shake flask. Production of SAKM3-L-K1 was induced by methanol (0.5% final concentration) administered every 12 h during the entire culture period. After 28–30 h of culture, the culture supernatant was collected by centrifuging the cells at 3,000 × g for 5 min and applied to a lysine-agarose column (Sigma) equilibrated with column binding buffer (50 mm Tris-HCl, 50 mm NaCl, pH 7.5). After washing the column with 3–5 bed volumes of the binding buffer, SAKM3-L-K1 was eluted with 0.15 mε-amino-N-caproic acid (EACA) (Sigma). Eluants were analyzed to determine yield and purity of SAKM3-L-K1 by SDS-PAGE and Coomassie Blue staining. The fractions selected were pooled, dialyzed against the binding buffer, and concentrated by ultrafiltration (Millipore Corp.). Purified SAKM3-L-K1 was quantified spectrophotometrically at 280 nm using a molar extinction coefficient of 35,350 m−1 cm−1 (35Gill S.C. Von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar). Purified SAKM3-L-K1 in deionized water was mixed with the matrix solution of sinapinic acid and analyzed on an Applied Biosystems Voyager-DE PRO mass spectrometer calibrated with trypsinogen (m/z 23,981). The instrument operated in the linear mode with an acquisition range 10,000–35,000 Da. This analysis was performed at the Alberta Peptide Institute, Edmonton, Alberta, Canada. Purified SAKM3-L-K1 was extensively dialyzed against 150 mm sodium phosphate, pH 7.4, and quantified spectrophotometrically at 280 nm as previously described. Titrations were performed using a Microcal VP-ITC calorimeter at 25 °C in 150 mm sodium phosphate, pH 7.4, following the manufacturer's guidelines and using Microcal Origin for data analysis. EACA (Sigma) was used as the ligand. The incremental heat change accompanying binding was corrected for the corresponding heat of dilution of EACA into buffer that was obtained in a separate experiment by titrating EACA into the sample cell containing buffer only. An ELISA method was used to assess the fibrin binding ability of SAKM3-L-K1 and SAK. Cross-linked fibrin was formed on the wells of a Nunc-Immuno MaxiSorp module (Nalge Nunc International Corp.) using the procedure described by Wu et al. (36Wu S.-C. Yeung J.C. Duan Y. Ye R. Szarka S.J. Habibi H.R. Wong S.-L. Appl. Environ. Microbiol. 2002; 68: 3261-3269Crossref PubMed Scopus (147) Google Scholar). Formation of cross-linked fibrin was confirmed by SDS-PAGE (36Wu S.-C. Yeung J.C. Duan Y. Ye R. Szarka S.J. Habibi H.R. Wong S.-L. Appl. Environ. Microbiol. 2002; 68: 3261-3269Crossref PubMed Scopus (147) Google Scholar). The fibrin was digested with plasmin (Roche Applied Science) at 4 milliunits/well (∼1 pmol/well) at room temperature. At different time points, the unbound plasmin was removed immediately by washing 4 times with PBST (0.1 m sodium phosphate, 0.15 m sodium chloride, pH 7.2, 0.1% Tween 20). Purified SAKM3-L-K1 or SAK in PBST containing 3% bovine serum albumin was added to the wells at a final concentration of 200 nm (20 pmol/well). After 2 h at room temperature, unbound materials were removed by washing. SAKM3-L-K1 or SAK retained on the well was probed with polyclonal antibodies against SAK (31Ye R. Kim J.H. Kim B.G. Szarka S. Sihota S. Wong S.-L. Biotechnol. Bioeng. 1999; 62: 87-96Crossref PubMed Google Scholar) followed by horseradish peroxidase-conjugated anti-mouse secondary antibodies. The amount of horseradish peroxidase retained was assessed using 1-Step Turbo TMB (Pierce) as the horseradish peroxidase substrate according to the manufacturer's instructions. Color development at end point was determined at 450 nm using a microplate reader (CERES 900, Bio-Tek Instruments, Inc.). The experiment was repeated three times. Fibrin clots were formed by adding human thrombin (to 0.6 NIH units/ml) (1NIH unit = 0.324 ± 0.073 μg thrombin) and CaCl2 (to 20 mm) to human fibrinogen (1 mg/ml, final concentration) in HEPES-buffered saline (HBS; 0.01 m HEPES, 0.13 m NaCl, pH 7.4). Both thrombin and fibrinogen were highly purified materials from Sigma. Immediately after mixing, 100-μl aliquots of the polymerizing fibrin solution were pipetted to the wells of a microtiter plate (Falcon 3912 flat-bottom polyvinyl chloride plate, BD Biosciences). Clot formation was allowed to proceed for 3 h at room temperature. The surface of the clots was washed with HBS, and excess fluid was carefully removed. A 100-μl solution containing freshly mixed human plasminogen (1.5 μm, Sigma) and varied concentrations of purified SAKM3-L-K1 or SAK in HBS was layered on each clot. The changes in clot turbidity with time were monitored by measuring changes in the absorbance at 405 nm at 25 °C using the CERES 900 microtiter plate reader operated in the kinetic mode. After 30 min the surface of each clot was gently washed with HBS three times to remove any unbound SAKM3-L-K1 or SAK. 100-μl aliquots of plasminogen (1.5 μm) in HBS were layered on the clots, and measurement was immediately resumed until all readings reached the low plateau, which was taken as completion of clot lysis. Duplicate wells were prepared for each concentration of SAKM3-L-K1 or SAK in each experiment, and the experiment was repeated three times. As a control, some clots were layered only with HBS but treated otherwise the same. Blood was drawn by venipuncture from healthy adult donors (who had taken no aspirin in the preceding 2 weeks) into 1/10 volume of buffered sodium citrate (129 mm). Platelet-poor plasma (PPP) was prepared from the pooled citrated blood by centrifugation at 1,500 × g for 15 min and frozen immediately at −20 °C in aliquots. All plasma clot experiments were performed within 1 week of blood collection. Clot perfusion using an optical flow cell was based on the model described by Hantgan et al. (37Hantgan R.R. Jerome W.G. Hursting M.J. Blood. 1998; 92: 2064-2074Crossref PubMed Google Scholar). Clotting was initiated by adding human thrombin (to 1.0 NIH units/ml) and CaCl2 (to 30 mm) to freshly thawed PPP. 200 μl of the polymerizing plasma was transferred to the flow cell (Hellma Cells, model 178.710-QS) to fill up the optical chamber (80 μl) and the inlet and outlet ports. Clotting was allowed to proceed for 3 h at room temperature. The clot was then perfused with PPP for 20 min at 20 μl/min, followed by perfusion with SAKM3-L-K1 or SAK (200 nm in PPP, 20 μl/min) with the flow rate controlled by a Bio-Rad peristaltic pump. The change in absorbance at 600 nm at 37 °C was continuously monitored using a Beckman DU-65 spectrophotometer (Beckman Instruments) operated with a Kinetics Soft-Pac module. The experiment was performed three times for both SAKM3-L-K1 and SAK. Vent DNA polymerase (New England BioLabs) was used for all DNA amplification reactions. The sequence of all PCR products was confirmed to be free of PCR errors by nucleotide sequencing performed at the University Core DNA and Protein Services, University of Calgary, Calgary, Alberta, Canada. Purification of SAK and specific activity determination of SAK and SAKM3-L-K1 followed the procedure described by Szarka et al. (19Szarka S. Sihota E. Habibi H.R. Wong S.-L. Appl. Environ. Microbiol. 1999; 65: 506-513Crossref PubMed Google Scholar). Plasminogen activation assay with the radial caseinolysis method was performed as described by Wong (38Wong S.-L. Ye R. Nathoo S. Appl. Environ. Microbiol. 1994; 60: 517-523Crossref PubMed Google Scholar). SAK-L-K1 (Fig.1B) was produced as a secretory protein in the culture supernatant using B. subtilis WB800, an eight extracellular protease deficient strain (36Wu S.-C. Yeung J.C. Duan Y. Ye R. Szarka S.J. Habibi H.R. Wong S.-L. Appl. Environ. Microbiol. 2002; 68: 3261-3269Crossref PubMed Scopus (147) Google Scholar), as the host. Radial caseinolysis study showed that SAK-L-K1 and SAK had similar activities for plasminogen activation. However, more than 95% of SAK-L-K1 was found in the flow-through fractions with a lysine-agarose column even though the sample had been extensively dialyzed to ensure that the Kringle domain in the fusion protein was not saturated with free lysine molecules present in the culture medium (data not shown). The observation suggested a defective Kringle-1 domain in the fusion protein. This result is not unexpected since the Kringle-1 domain contains three pairs of disulfide bonds arranged in a 1–6, 2–4, 3–5 pattern (i.e. cysteine residues forming disulfide bonds are not arranged in a sequential manner, Fig.1B). Inability to form disulfide bonds or mis-pairing of cysteine residues can result in the formation of defective Kringle-1 domains (39Trexler M. Patthy L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2457-2461Crossref PubMed Scopus (67) Google Scholar). Because functional human plasminogen Kringle-1 domain has been shown to be produced efficiently from P. pastoris (40Zajicek J. Chang Y. Castellino F.J. J. Mol. Biol. 2000; 301: 333-347Crossref PubMed Scopus (14) Google Scholar), it would be a logical approach to produce SAK-L-K1 in P. pastoris. However, using P. pastoris as the production host of the SAK-L-K1 fusion protein has another concern. P. pastoris has been shown to produce SAK only in a partially active form because of an N-linked glycosylation at Asn-28 of the mature SAK (41Miele R.G. Prorok M. Costa V.A. Castellino F.J. J. Biol. Chem. 1999; 274: 7769-7776Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). To eliminate glycosylation of SAK-L-K1 in P. pastoris, two key residues (Table I, shown in bold) that constitute part of the consensusN-linked glycosylation site (Asn-28–Val-29–Thr-30, numbering according to the mature SAK sequence) were changed (Table I). The first two muteins, SAKM1-L-K1 and SAKM2-L-K1, have Asn-28 changed to Asp and Ala, respectively, whereas the third mutein SAKM3-L-K1 has Thr-30 changed to Ala. To quickly examine the effects of these mutations on SAK activity, the muteins were first produced in B. subtilis via secretion. Although the production yields of these muteins and the wild-type control, SAK-L-K1, from B. subtilis were comparable, radial caseinolysis study indicated that SAKM1-L-K1, SAKM2-L-K1, and SAKM3-L-K1 retained 1 (or less), 40, and 90% of the wild type SAK activity, respectively (data not shown). Structural genes encoding SAKM2-L-K1 and SAKM3-L-K1 were then transferred to the P. pastoris expression vector for further characterization. Secretory production of
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