Glycosylation of Asparagine-28 of Recombinant Staphylokinase with High-Mannose-type Oligosaccharides Results in a Protein with Highly Attenuated Plasminogen Activator Activity

Miele, Robert G.; Prorok, Mary; Costa, Vincent A.; Castellino, Francis J.

doi:10.1074/jbc.274.12.7769

Cited by 22 publications

(25 citation statements)

References 24 publications

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“…SAK gene has a single site for N-glycosylation at Asn 28 residue. When expressed in P. pastoris, the protein is glycosylated at this site (Miele et al 1999). Tunicamycin, castanospermine, glucosamine, PNGaseF, bacitracin, nisin, EDTA are N-linked glycosylation inhibitors (Luczak et al 2008;SebbanKreuzer et al 2006;Wang et al 2001;Bayley et al 1993).…”

Section: Glycosylated and Non-glycosylated Rsak Expressionmentioning

confidence: 96%

“…SAK gene has been cloned and expressed to varied levels in different expression systems like Escherichia coli, Bacillus subtilis, Streptomyces lividans, and Pichia pastoris (Nagnath et al 2009;Ren et al 2008;Ye et al 1999;Cheng et al 1998;Miele et al 1999).…”

Section: Introductionmentioning

confidence: 99%

“…The amino acid sequence of SAK has a potential N-linked glycosylation site at Asn 28 which when glycosylated is detrimental for its hPg activator activity (Miele et al 1999). To achieve a hyperexpressing, non-glycosylated, active rSAK in P. pastoris, we integrated the SAK gene into the Pichia genome; induction was then carried out with methanol in the presence or absence of tunicamycin.…”

Section: Introductionmentioning

confidence: 99%

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High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris

et al. 2009

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Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (approximately 1 g/l) of extracellular glycosylated rSAK (approximately 18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (approximately 15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.

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Section: Glycosylated and Non-glycosylated Rsak Expressionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris

et al. 2009

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“…DISCUSSION An important genetic manipulation in this study involves the production of properly folded, non-glycosylated SAK-Kringle-1 fusion protein in P. pastoris. Glycosylation of SAK in P. pastoris has been found to result in a SAK with attenuated plasminogen activator activity (41). The presence of the oligosaccharide moiety is suggested to cause subtle changes in the orientation of plasmin so that the complex is less optimal in plasminogen activation.…”

Section: Plasma Clot Lysis Kinetics: Effects Of Sakm3-l-k1mentioning

confidence: 99%

“…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 (41). To eliminate glycosylation of SAK-L-K1 in P. pastoris, two key residues (Table I, shown in bold) that constitute part of the consensus N-linked glycosylation site (Asn-28 -Val-29 -Thr-30, numbering according to the mature SAK sequence) were changed ( Table I).…”

Section: Construction Of B Subtilis Expression Vectors For Sak-l-k1 mentioning

confidence: 99%

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

Wong

2003

Journal of Biological Chemistry

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To develop a fast-acting clot dissolving agent, a clottargeting 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 in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfidebond 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 flowcell 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.

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Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Faraji

Ramezani

Mashkani

et al. 2019

Biotechnology Progress

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Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood‐clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20‐amino acid linker with 2 RGD (2 × arginine‐glycine‐aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72‐hr stimulation with 2.5% methanol and remained steady for 3–4 days. The highest expression was obtained at the range of 2–3% methanol. The SAK‐2RGD‐TT (relative activity >82%) was more active at 25–37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7–9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK‐2RGD‐TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK‐2RGD‐TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK‐2RGD‐TTI with high activity without requiring a complicated purification procedure.

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Glycosylation of Asparagine-28 of Recombinant Staphylokinase with High-Mannose-type Oligosaccharides Results in a Protein with Highly Attenuated Plasminogen Activator Activity

Cited by 22 publications

References 24 publications

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

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