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PandaPure is a technology to transform host cells (e.g. bacterial E. coli ) into a machinery to process and purify the recombinant proteins it expresses.
In order to launch PandaPure system, you would need the following key components.
DNA molecule to transform host to build a synthetic organelle with cognate receptors to sort the protein of interest
DNA molecule that encodes the coding sequence of a fusion of your protein of interest (POI) and an signal tag to address synthetic organelles.
To cleave signal tags from POI, thus release POI from synthetic organelles, enabling protein purifications.
Cell culture for DNA-encoded program to work, including host cells, inducers, growth medium.
Reagents to lyse harvested cells, to release cytosolic impurities. Multiple options are available.
How to make your PandaPure Constructs? We offer flexible options with PandaPure line-up.
We designed PandaPure®️ Protein Construct as a two-plasmid system encoding organelle and protein expression separately (Table 1).
An optimized version PandaPure Organelle Plasmid is provided in all product and services.
The expression plasmid can be made either by in-house cloning with kits, or out-sourcing.
Clone by yourself using PandaPure®️ kits
Obtain the best flexibility to integrate PandaPure into your own workflows.
Available at: PandaPure kits
Outsource to PandaPure®️ Construct Service
Unlock the potential of PandaPure with our tailored DNA construct services. Simply submit your protein of interest.
Available at: PandaPure service
About the control construct
One of the most common expression vector for traditional expressin and affinity purification is pET-28a(+) from Novagen, where His-tag can be used for both purification and Western blot.
Here we use it as a control. In the event that proteins fail to express with PandaPure, we can use this control construct to evaluate whether protein is difficult or not compatible with PandaPure.
PandaPure®️ kits contains pOrganelle, PandaPure Organelle Plasmid, and pPPC-vector, PandaPure Expression Vector.
To make your own PandaPure Expression Plasmid: inserting coding sequence (CDS) of your protein into pPPC-vector, by homolog-based cloning methods (e.g. Gibson assembly).
PandaPure®️ Construct Service offers an end-to-end process from sequence optimization, gene synthesis, cloning, and plasmid preparation. You just need to submit your proteins of interest, by providing amino acid sequence, or DNA, or Uniprot ID. Then you will receive ready-to-use plasmids in a few weeks depending on the complexity of the genes, including:
- Ready-to-use PandaPure Protein Construct
- Control Construct: Cloning your genes in pET-28a(+)
We offer necessary reagents for PandaPure, included in predefined kits, and also available as individual components and in the services.
PandaPure®️ Protein Reagent
All-in-one reagent for tag cleavage, removal and protein purification. The key reagent of PandaPure workflow.
PandaPure®️ Lysis Reagent
Gentle disruption of bacteria e.g. E. coli in 15 minutes, based on nonionic detergent.
Notably, other nonionic detergents can also be used for PandaPure. Here are some tested alternatives.
a commonly used nonionic detergent
Description: PandaPure Protein Reagent enables efficient and precise cleavage of signal tag encoded in PandaPure Expression Plasmid, and initiate protein release and purification. Buffers are compatible with both manual and automated applications with liquid-handling workstations, for seamless protein purification powered by PandaPure technologies. Depending on the particular application, additional components, such as protease inhibitors, salts, and reducing agent may be added to the reagent.
Capacity: Up to 30 mg proteins per 1 mL reagent
Buffer: 0.1 mM HEPES, pH 8.2
Store: at room temperature.
Description: PandaPure®️ Lysis Reagent enables gentle lysis of bacteria (E. coli) without the mechanical disruption. The reagent is also compatible for other methods for protein extraction and purification from bacterial cell lysates. Lysozyme and DNase I can be further supplied to improve extraction efficiency of large proteins (MW > 70kDa).
Capacity: Up to 1 g bacteria per 8 mL reagent
Buffer: Tris-HCl, pH 7.5
Store: 4°C or -20°C (long term)
Is expression success guaranteed by PandaPure?
PandaPure is designed to enhance protein production and often performs better than traditional E. coli systems.
But, it's important to acknowledge that biological processes are inherently complex and subject to many variables, some of which are still not fully understood.
We cannot guarantee successful protein production in every case.
Can PandaPure also work for traditional vectors?
They cannot, unfortunately. PandaPure technology requires the host cells to do specific works intracellularly, which are encoded in sequences from PandaPure Protein Construct, and not available from other commercial systems.
But they will make a great comparison to PandaPure! To apply PandaPure, you can order PandaPure Kit, PCR your genes, and cloned it into PandaPure expression vector.
You can clone your gene into the pET28a(+) vector as a control, (available through our PandaPure Construct Service).
If a protein fails to express in both PandaPure and pET28a(+), it may require further protein engineering, for such cases, through Ailurus Protein Service for AI-aided, high-throughput screening. If your gene doesn't express in PandaPure but does in pET28a(+), you can switch to the traditional system.
For detailed troubleshooting, please refer to the detailed guidance provided in our User Guide document. If you encounter issues, our support team is also available to help you optimize your experiments.
pPPC-vector can be linearized by a simple NcoI restriction enzyme digestion.
Then you can apply Gibson Assembly or other types of long-homology-based cloning method, with the following overlapping regions.
PandaPure technology have been tested in many lab strains of E. coli.
Our current offering uses T7 promoters to drive gene expression, thereby requiring T7 polymerase-encoding hosts, such as BL21(DE3) and BL21AI.
Also, our offerings contain kanamycin (Kan) and chloramphenicol (Cm) resistances, and cannot be selected in hosts with the same markers.
For needs in other hosts, please contact us for assistance at: support@ailurus.bio
Note: MG1655, and DH10B are tested when target proteins are expressed under E. coli native promoters. T7 RNAP needs to be integrated if applying our current offerings.
Our current offering has been tested from microliters in deep-well plates, to liters in bench-scale bioreactors.
For research use, we recommend 5 ml culture for preliminary trial, and 50 ml or 250 ml culture for scale.
Notably, as it uses isopropyl β-D-1-thiogalactopyranoside (IPTG) and anhydrotetracycline (aTc) for induction, two antibiotics for selection, and detergent for cell lysis, current offering and protocols are not designed for larger scale and may not be economical.
If you want to scale up your protein production with PandaPure, please contact us for assistances at: support@ailurus.bio
PandaPure is compatible with many different conditions
For bacterial growth, you can use shaking incubator for microplates, tubes, or flasks, and also bioreactors.
For separation, you can apply centrifugation. You may need plate centrifuges for microplate, or bench-top centrifuges for tubes from 1 ml to 50 ml, or large ones for higher volumes at 4,000 -16,000 g for efficient separation.
Other techniques (e.g. TFF) can also be used for PandaPure, as long as they are efficient method to separate materials at molecular scale (nanometer) and micrometer scale.
For users of PandaPure toolkits, you may also need full equipment for molecular biology, e.g. thermocycler or PCR machine.
Why two plasmids?
PandaPure does work in a single-plasmid system or with even more plasmids.
However, as LLPS molecules that form organelles are often highly repetitive, from our experience, the best way is to encode all organelle components in one plasmid, away from POI to avoid context effects.
We offer genetically stable Organelle Plasmid as a on-shelf product.
DISCLAIMER: if you choose to design your own constructs and encounter any issues, we cannot assume responsibility for these problems, as this usage falls outside our recommended guidelines and intended use of the product.
Documentation
PandaPure Workflow: Protein Purification
with 0 column, 0 beads, 0 protease
