ET SSB PyroPozzi®
PyroPozzi® is an extremely thermostable single-stranded DNA binding protein (ET SSB) isolated from a hyperthermophilic microorganism near Pozzuoli, Italy. The protein has been improved by directed evolution and rational design and remains fully active after incubation at 100°C for 12 hours. Due to its extreme thermostability, PyroPozzi® can be used in applications that require extremely high-temperature conditions, such as nucleic acid amplification by PCR and Sanger sequencing.
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FAQs & Troubleshooting
- Improve the processivity of DNA polymerase
- Stabilization and marking of ssDNA structure
- Increase the yield and specificity of PCR reactions
- Increase the yield and processivity of RT during RTPCR
- Improve DNA sequencing through regions with strong secondary structure
- Enhance the RecA activity for ssDNA binding and strand transfer
PyroPozzi® is active in most PCR buffers employing family A, e.g. Taq, or family B, e.g. Pfu/KOD, DNA polymerases at a final concentration of 4 ng/µl (~100 ng for a 25 µl PCR reaction).
PyroPozzi® single stranded binding (SSB) protein variant was improved through protein engineering efforts including directed evolution and rational design. The final engineered protein was synthesized as a codon-optimized minigene and expressed heterologously in an E. coli strain. The recombinant protein was tested and normalized to standard activity assays.
>95 % by SDS-PAGE and analysis by protein mass spectrometry
Purified recombinant protein was subjected to peptide mass fingerprinting (PMF) by MALDI-TOF/TOF (PMF + MS/MS).
The protein is sold by mass as determined by UV-spectroscopy (OD280nm) and ESI-TOF mass spectrometry and tested to be free of endonuclease and exonuclease activities.
Purified PyroPozzi® is tested in a challenging multiplex PCR at a final concentration of 4 ng/µl to evaluate the effect of SSB proteins on PCR specificity and compared to non-spiked SSB multiplex PCR via DNA polyacrylamide gel electrophoresis (DNA PAGE) in low conductivity lithium borate acetate media. Additionally, purified PyroPozzi® is assayed for its specificity and efficiency in amplifying multiple long fragment targets within the human genome in long-range PCR assays and, likewise, compared to non-spiked SSB long-range PCR reactions. The long amplicons are separated by pulsed-field gel electrophoresis (PFGE) or field inversion gel electrophoresis (FIGE) in 1% agarose gel containing a TBE buffer and compared to a double-stranded DNA molecular weight marker.
Lipopolysaccharide (LPS) was determined by the standard LAL (Limulus amebocyte lysate) test to be < 1 EU/µg. Due to potential environmental sustainability concerns (collection of the hemolymph used in pharmaceutical testing may negatively affect horseshoe crab populations), a quantitative and more sensitive endotoxin assay was developed using a pyrogen‐testing cell model with knock-in (targeted, single-copy integration) of TLR4/CD14/MD2 at the safe harbor locus AAVS1/PPP1R12C on human chromosome 19. The TLR4/CD14/MD2 assay is used as an orthogonal screen for LPS in lieu of the LAL method and has a detection limit of 0.005 EU/ml.
A sterile, aqueous, clear and colorless solution.
Storage & Stability:
The product is shipped at ambient temperature. Upon receipt, store it immediately at -20°C.
PyroPozzi® is supplied at 1 mg/ml in stabilization buffer and remains bioactive for 30 days if maintained refrigerated [2º to 8ºC (35º to 46ºF)]. Stock solutions of PyroPozzi® in its concentrated form can be stored at least 9 months at -20°C and at least 18 months at -80°C from the date of receipt with no loss of activity. Avoid repeated freeze-thaw cycles by aliquoting. Multiple freeze-thaw cycles reduce potency and, therefore, aliquoting working stocks is strongly recommended.
PyroPozzi® is supplied reconstituted at 1 mg/ml in a stabilization buffer containing 50 mM Tris-HCl, 100 mM potassium chloride, 100 mM ammonium sulfate, 1% w/v TritonX-100, 50% v/v glycerol at a pH of approximately 7.9. The reconstituted solution has been filter sterilized by passing through a 0.22 micron PES membrane and tested to be negative for mycoplasma contamination.
Disclaimer & Precautions:
This product is solely for research and development use only and may be subject to conditional use and licensing restrictions. The product shall not be used as an advanced pharmaceutical intermediate (API) or investigational drug or a biologic. This product is not intended to be used as a therapeutic agent or facilitate clinical diagnosis or be used as an in vitro diagnostic (IVD) product.
The Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER) define an IVD as: “In vitro diagnostic products are those reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended for use in the collection, preparation, and examination of specimens taken from the human body. These products are devices as defined in section 201(h)of the Federal Food, Drug, and Cosmetic Act (the act), and may also be biological products subject to section 351 of the Public Health Service Act. Title 21, Code of Federal Regulations (CFR), section 809.3(a).”
This product may not be used or formulated in any agricultural, pesticidal, veterinary or animal products, food additives or household chemicals or any other unspecified use. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. NovoHelix distributes products for basic and translational research use only.
Notice to purchaser:
The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents or applications owned by NovoHelix to use this product. No other license under these patents or applications is conveyed expressly or by implication by purchase of this product.
FAQs & Troubleshooting
1: DiDonato M, Krishna SS, Schwarzenbacher R, McMullan D, Jaroszewski L, Miller MD, Abdubek P, Agarwalla S, Ambing E, Axelrod H, Biorac T, Chiu HJ, Deacon AM, Elsliger MA, Feuerhelm J, Godzik A, Grittini C, Grzechnik SK, Hale J, Hampton E, Haugen J, Hornsby M, Klock HE, Knuth MW, Koesema E, Kreusch A, Kuhn P, Lesley SA, Moy K, Nigoghossian E, Okach L, Paulsen J, Quijano K, Reyes R, Rife C, Spraggon G, Stevens RC, van den Bedem H, Velasquez J, White A, Wolf G, Xu Q, Hodgson KO, Wooley J, Wilson IA. Crystal structure of a single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60 A resolution. Proteins. 2006 Apr 1;63(1):256-60. PubMed PMID: 16435371.
1: Rapley R. Enhancing PCR amplification and sequencing using DNA-binding proteins. Mol Biotechnol. 1994 Dec;2(3):295-8. PubMed PMID: 7866882.
1: Olszewski M, Grot A, Wojciechowski M, Nowak M, Mickiewicz M, Kur J. Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana. BMC Microbiol. 2010 Oct 15;10:260. doi: 10.1186/1471-2180-10-260. PubMed PMID: 20950419; PubMed Central PMCID: PMC2964679.
1: Olszewski M, Rebała K, Szczerkowska Z, Kur J. Application of SSB-like protein from Thermus aquaticus in multiplex PCR of human Y-STR markers identification. Mol Cell Probes. 2005 Jun;19(3):203-5. PubMed PMID: 15797821.
1: Wang W, Sun L, Zhang S, Zhang H, Shi J, Xu T, Li K. Analysis and prediction of single-stranded and double-stranded DNA binding proteins based on protein sequences. BMC Bioinformatics. 2017 Jun 12;18(1):300. doi: 10.1186/s12859-017-1715-8. PubMed PMID: 28606086; PubMed Central PMCID: PMC5469069.
1: Ali F, Arif M, Khan ZU, Kabir M, Ahmed S, Yu DJ. SDBP-Pred: Prediction of single-stranded and double-stranded DNA-binding proteins by extending consensus sequence and K-segmentation strategies into PSSM. Anal Biochem. 2019 Nov 3;589:113494. doi: 10.1016/j.ab.2019.113494. [Epub ahead of print] PubMed PMID: 31693872.
1: Nanni L, Brahnam S. Set of approaches based on 3D structure and position specific-scoring matrix for predicting DNA-binding proteins. Bioinformatics. 2019 Jun 1;35(11):1844-1851. doi: 10.1093/bioinformatics/bty912. PubMed PMID: 30395157.