Secretory Production and Purification of Functional Full-Length Streptavidin from Bacillus subtilis

Wu, Sau‐Ching; Qureshi, Mohammad Hassan; Wong, Sui‐Lam

doi:10.1006/prep.2001.1582

Cited by 33 publications

(21 citation statements)

References 28 publications

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“…Production and Purification of Streptavidin-Wild-type streptavidin was produced by B. subtilis WB800(pSSAV-Tcry) cultured in a defined medium (14). The secreted protein was purified to homogeneity using cation exchange followed by iminobiotin affinity chromatography (12).…”

Section: Construction Of Streptavidinmentioning

confidence: 99%

“…In combination with L109T mutation, a series of single, double, and quadruple streptavidin muteins were created and produced from Bacillus subtilis via secretion. As they were produced in the soluble form without the requirement of refolding (13,14), their oligomeric state can be rapidly analyzed by SDS-PAGE using culture supernatants from the streptavidin mutein production strains. The muteins were purified and further characterized by different approaches to confirm their monomeric state.…”

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confidence: 99%

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Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability

Wong

2005

Journal of Biological Chemistry

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Monomeric streptavidin with reversible biotin binding capability has many potential applications. Because a complete biotin binding site in each streptavidin subunit requires the contribution of tryptophan 120 from a neighboring subunit, monomerization of the natural tetrameric streptavidin can generate streptavidin with reduced biotin binding affinity. Three residues, valine 55, threonine 76, and valine 125, were changed to either arginine or threonine to create electrostatic repulsion and steric hindrance at the interfaces. The double mutation (T76R,V125R) was highly effective to monomerize streptavidin. Because interfacial hydrophobic residues are exposed to solvent once tetrameric streptavidin is converted to the monomeric state, a quadruple mutein (T76R,V125R,V55T,L109T) was developed. The first two mutations are for monomerization, whereas the last two mutations aim to improve hydrophilicity at the interface to minimize aggregation. Monomerization was confirmed by four different approaches including gel filtration, dynamic light scattering, sensitivity to proteinase K, and chemical cross-linking. The quadruple mutein remained in the monomeric state at a concentration greater than 2 mg/ml. Its kinetic parameters for interaction with biotin suggest excellent reversible biotin binding capability, which enables the mutein to be easily purified on the biotin-agarose matrix. Another mutein (D61A,W120K) was developed based on two mutations that have been shown to be effective in monomerizing avidin. This streptavidin mutein was oligomeric in nature. This illustrates the importance in selecting the appropriate residues and approaches for effective monomerization of streptavidin.(Strept)avidin with reversible biotin binding capability can extend the applications of the biotin-(strept)avidin technology. These molecules can be applied for affinity purification of biotinylated biomolecules, screening of ultratight binders binding to biotinylated biomolecules displayed on the phage display system, and development of reusable biosensor chips, protein/ antibody microarrays, and enzyme bioreactors (1, 2). (Strept)avidin is a homotetrameric molecule with a biotin binding site in each subunit (3). The three-dimensional structure of (strept)avidin (4, 5) suggests that a complete biotin binding pocket in each subunit requires the contribution of a tryptophan residue from an adjacent subunit. Site-directed mutagenesis studies also demonstrate the importance of this residue for tight biotin binding and subunit communications (6 -8). Therefore, development of monomeric (strept)avidin can be an attractive approach to engineer (strept)avidin with reversible biotin binding capability.The engineering of (strept)avidin to its monomeric form is technically challenging. In the case of avidin, the first generation of engineered monomeric avidin can exist in the monomeric state only in the absence of biotin (9). This problem has been solved by the recent development of the second generation of monomeric avidin (10), which carries two mut...

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Section: Construction Of Streptavidinmentioning

confidence: 99%

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confidence: 99%

Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability

Wong

2005

Journal of Biological Chemistry

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“…Bacillus subtilis WB700 was kindly provided by Dr. Sui-Lam Wong [15]. Escherichia coli DH5a and Bacillus megaterium ATCC14945 were purchased from Novagen (Darmstadt, Germany) and America typical culture center (ATCC), respectively.…”

Section: Bacterial Strains Growth Conditions and Primersmentioning

confidence: 99%

Development and Application of a Novel Signal Peptide Probe Vector with PGA as Reporter in Bacillus subtilis WB700: Twenty-Four Tat Pathway Signal Peptides from Bacillus subtilis were Monitored

Zhu

Zhang³

et al. 2008

Mol Biotechnol

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In this study, we have developed a novel, versatile signal peptide probe vector driven by promoter P43 in Bacillus subtilis WB700, using Penicillin G Acylase (PGA) as reporter. Twenty-four signal peptides considered belonging to twin-arginine translocation (Tat) pathway were cloned into the probe vector to direct the secretion expression of PGA, respectively. Through 6-nitro-3-phenylacetamidobenzoic acid (NIPAB) filter paper assay, four signal peptides (AmyX, AlbB, LipA, and YmzC) were chosen for further investigation. The extracellular production of PGA demonstrated that these recombinants mediated efficient secretion expression in B. subtilis WB700, in which the maximum activity reached 0.11, 0.21, 0.08, and 0.26 U/mL, respectively. Thus, we provided an efficient tool for easy detection of the signal peptides in B. subtilis, and demonstrated the efficiency of Tat pathway signal peptides via PGA secretion in B. subtilis WB700.

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“…The structural genes encoding both K coil and E coil sequences were assembled by using several long synthetic oligonucleotides (primers 1-4 for K coil and primers 5-8 for E coil) with a PCR-based strategy (22,23). For the K coil sequence, portions of the primer 2 sequence are complementary to primers 1 and 3, respectively, and part of the primer 4 sequence is complementary to primer 3.…”

Section: Assembly Of K Coil and E Coil Sequences-mentioning

confidence: 99%

Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots

Lian¹,

Szarka²,

et al. 2003

Journal of Biological Chemistry

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Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.

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Secretory Production and Purification of Functional Full-Length Streptavidin from Bacillus subtilis

Cited by 33 publications

References 28 publications

Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability

Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability

Development and Application of a Novel Signal Peptide Probe Vector with PGA as Reporter in Bacillus subtilis WB700: Twenty-Four Tat Pathway Signal Peptides from Bacillus subtilis were Monitored

Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots

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