Big Potential in Using Protozoans for Producing Mammalian Proteins

Recombinant protein expression is critical for functionally studying proteins, preparing antigens, providing tissue culture growth supplement, and producing certain therapeutic compounds. Like many molecular biology labs, we have used several heterologous protein expression systems over the last decade including E. coli, yeasts, insect cells and mammalian cells from various species. It is widely accepted that these systems present increasing functional relevance from bacteria to mammalian cells, with accompanying increase in difficulty and cost. The benefits of using cells from higher species are often reflected in post-translational modifications (PTMs), such as glycosylation, phosphorylation, etc.

There is yet another system that could be easy to handle while maintaining mammalian-like PTMs–parasitic protozoan Leishmania tarentolae. L. tarenolae is a unicellular organism, its host is lizard. Even though it’s a vertebrate parasite, this species poses no risk to humans. Amazingly, L. tarenolae individuals can be grown on agar plates for clonal selection or in simple liquid media like E. coli. Their optimal growth temperature is 27C, and they do not require shaking; thus they are suitable for growth in insect cell incubators or even at room temperature. The most important advantage of this system is that oligosaccharide structures of proteins produced in this organism resemble those of mammalian cells much more closely than even insect cells, i. e. the N-glycosylation profile can be basically identical to a biantennary fully galactosylated Man3GlcNAc2core-a-1,6-fucosylated structure found in mammalian cells.

IFrom our first-hand experience, the handling of this species is extremely convenient. While we heavily promote the baculovirus expression system (BVES) for most of our custom protein production projects (we carried out one NIH project for producing human glycosylated cancer antigen proteins using a modified BVES recently), we now believe that there is a good chance that many of the proteins we have been producing could be produced in the protozoan system with potentially better efficiency.

New Product of the Week: GFP-Multitrap 5 plates, ABP-CM-GMULT5, $1,200.

Promotion of the week: Get one GFP-Trap free when you send us two referrals. Call 858-587-6645 for details or claim prize.

Tags: BVES, glycosylation, insect cells, L. tarenolae, Leishmania tarentolae, Lexsy, protein epxression, protein yield, protozoan, PTM, recombinant proteins

Our New Website is Complete, 10% off this week

We recently launched our new website, and to celebrate the launch, we’ll be offering a discount of 10 % on all our products and services from July 18th to July 22nd! Come explore a wide variety of products that Allele Biotech has to offer from viral expression to fluorescent proteins. Examples of services offered include custom lentiviral, retroviral, and baculoviral packaging along with cell production and cell line development. This is our effort to enhance your online shopping experience through our improved shopping cart system. Our mission remains the same; to increase accessibility to innovative molecular biology research tools by offering cutting edge products at a reasonable cost. Please visit http://www.allelebiotech.com and use the code NEWSITE to redeem the offer. We thank you for your support!
For more details click here

Tags: Fluorescent proteins, iPS, Online Shopping, RNAi, viral packaging

New Frontiers for Research Tool Development in the New Year

Allele Biotech's Green Crystal Ball

Optogenetics
Chosen as the Method of the Year 2010 by Nature Method and mentioned in a number of year-end recaps, this is a technology that allows the use of light to precisely (at least in a temporal sense) control engineered proteins within a targeted cell population. For example, by introducing light-activated channelrhodopsins into neurons, one can use a pulse of light to initiate a movement of ion across the cell membrane. The technology, first reported in 2005 then made headlines as a major impact on neurosciences since 2007, is now being combined with other components in controlling a broader array of biological events, such as DNA binding, enzyme activities, etc. Looking forward, a few areas will be more than likely the frontlines of moving optogenetics into more labs:

Additional combinations: The few known channelrhodopsins and their fast growing variations will be combined with more “effecter” domains to control different events. The challenge will be to find ways to use the structural changes or any responses channelrhodopsins have to stimulating lights in order to trigger a reaction in the associated effecter domain.

Tracking mechanisms: A platter of fluorescent proteins (FPs) will be used as an independent tracking method to follow cells being targeted. FPs that have optical spectra that do not interfere with the optogenetic molecules will be tested and established. In addition, FPs with less toxicity, narrower excitation and emission peaks, and more tolerance to different cellular environment will be preferred and eventually set up as standards.

Delivery tools: To bring the optogenetic reagents into cells like neurons researchers will most likely rely on lentiviral vectors in most cases. Other vehicles such as baculovirus, MMLV-based retrovirus, even herpes virus may find broader applications in this field. Pre-packaged lentiviruses and MMLV-retroviruses already contain optogenetic constructs will become popular products.

VHH Antibodies
The small capture polypeptides based on single-domain Camelid antibodies (nanobodies, nano antbodies or nAbs) and similar VHH domains will become much dramatically more popular this year, judging from the significant increase in demands of the only camelid reagent products, GFP-Trap and RFP-Trap, in 2010. There are a number of NIH initiated programs that aim to find capture reagents that eventually target the complete human proteome. One of the key criteria for the current phase of the relevant NIH Director’s Initiative is ability to co-immunoprecipitate. The Human Proteome Organization (HUPO) recently expressed frustration due to the lack of high quality capture reagents necessary to isolate and identify most proteins. HUPO promotes global research on proteins in order to decode the human proteome. From what we have learned from dozens of publications showing the use of GFP-Trap, VHH molecules pulls down GFP-tagged proteins with unprecedented efficiency and purity. VHH antibodies show strong affinity and specificity, at a level superior or comparable to monoclonal antibodies. In addition, VHH antibodies are increasingly appreciated for their capabilities to recognize concave epitopes by their relatively convex-shaped paratopes. VHH nanobodies are small (~12-15 kD), with a limited number of functionally important disulfide bonds, can be expressed very well in E. coli, and are amazingly stable in extreme denaturing conditions such as heat and acid. They have been shown to be better suited for in vivo and trans-cellular membrane delivery than other antibodies. It should not be surprising that one day in the coming years VHH antibodies will be more dominant than monoclonal antibodies.

Super-Resolution Imaging
One of the goals of developing technologies such as photoactivated localization microscopy (PALM) and related super-resolution imaging (SRI) techniques was to achieve electron microscopy (EM) level resolution without using EM. Now new developments show that maybe combining EM and photoactivable FPs would provide more specific and more detailed morphology. It would be anticipated that more photoconvertible FPs will prove to work well for one type of SRI or another. The event that will bring this technology to nearly every cell biology lab is the improvement and availability of necessary instruments that some companies have already begun to commercialize.

New Product of the Week 010311-010911:

Human let-7b miRNA minigene on lentivirus with RFP reporter, ABP-RP-MILT7BLP

Promotion of the Week 010311-010911:

15% off mWasabi-based organelle markers carried on baculo2mammalian system if order this week (On-Demand products will require about 3-4 weeks for virus packaging after an order is placed). Use code 0103BACFP on fax or email order.

Tags: alpaca antibodies, camelid antibodies, channelrhodopsin, EM, halorhodopsin, optogenetic, PALM, photoactivable FP, photoconvertible FP, STED, STORM, VHH, VHH antibodies

Working on the Wnt signaling pathway

Wnt proteins form a family of highly conserved, secreted glycolipoproteins that regulate cell proliferation, cell polarity and cell fate determination during embryonic development and tissue homeostasis. Mutations in Wnt genes or the Wnt signaling pathway components are often linked to human birth defects, cancer and other diseases.

Since the discovery of the Wnt-1 gene 27 years ago, a complex Wnt signaling network with many components having multiple distinct roles and acting in different cellular compartments has been established. The studies of Wnt signaling in human diseases, in stem cell biology and tissue regeneration holds promise for translational medicine.

A few commonly asked questions regarding studies on the Wnt pathways will be touched upon in this blog series:

How to activate the Wnt Signaling Pathway?

1) Recombinant Wnt Proteins: there are several commercial sources for recombinant Wnt proteins. As most of them are produced in bacteria, endotoxin, purity and active concentration are of primary concern. From our own experience using commonly used commercial Wnt proteins, the activities vary from batch-to-batch, and sometimes the products are simply inadequate.

2) Conditioned Medium: this approach is convenient and of low cost. ATCC provides two mouse fibroblast cell lines, which over-express mouse Wnt3A (CRL-2647) and mouse Wnt5A (CRL-2814). Researchers can maintain the cell lines and collect conditioned medium whenever they need Wnt proteins. However, these are the only two Wnt over-expressing cell lines available on the market, yet there are 19 members in the Wnt gene family. If you are planning on working with Wnt proteins other than 3A and 5A, you would have to develop relevant cell lines on your own. On the other hand, because over-expression of Wnt proteins will also activate the secretion of some growth factors, such as FGFs, the conditioned medium should be a mixture of several kinds of secreted factors.

3) Over-expression Wnt proteins directly in your cells of interests: Over-expression is a basic strategy for functional biology research. One of the barriers for introducing cDNAs into cells is the transfection efficiency. The advent of retroviral transfection (MMLV based or Lenti based) technically resolves this problem, as the transduction efficiency can reach almost 100%. However, highly efficient retrovirus packaging remains a difficult process for most research labs.

Allele Biotech plans to introduce all the human Wnt cDNAs on its HiTiter™ Expression Lentivirus platform as shelf products. The package size will be 5 vials, each vial 100 µl, the titer is 10e8 TU/ml at a price of $799. This product line will be under “Product-on-Demand”, with clones ready but packaged only upon order for the first time. First-time orders will qualify for a 10% discount, if the first-time customer provides the cDNA, there will be an additional 20% discount. These lentiviruses can be used directly in target cells or to build over-expressing cell lines for conditioned medium. Next topic is How to inhibit Wnt signaling pathway?

New Product of the Week 120610-121310: Human miRNA minigene on lentivirus with RFP reporter, ABP-RP-MI221LP

Promotion of the Wee 120610-121310: 5% off virus packaging if you use our FaceBook code “ViralPackaging” at shop.allelebiotech.com (beta-test new web).

Tags: cell fate, cell polarity, cell proliferation, conditioned medium, growth factors, lentivirus, signaling pathways, Wnt

How to order virus packaging service

Step 1: Please review the following before ordering Allele’s retrovirus packaging service.

Verify that your plasmids for virus production meet the following criteria:

No internal polyA signal or polyA signal immediately downstream of the coding cistron.

Avoid toxic genes, unusual secondary structures, and expression cassettes > 8kb (5’ to 3’ LTR).

If these concerns can not be satisfied in your vector design, please contact our technical specialist (vivec@allelebiotech.com) for further discussion, as Allele’s proprietary packaging technology might be able to help.

Step 2: Select the service:

Allele offers multiple project discounts: a 5% discount for ordering 3-5 packaging services, and a 10% for 6-10 packaging services.

Additional information can be found here.

Step 3: Send us 10 ug of endotoxin-free plasmid (blue ice shipping) for virus packaging.

To facilitate the completion of the custom virus packaging projects, Allele offers a variety of retro/lentivirus plasmids as well as viral plasmid subcloning and endotoxin-free plasmid preparation services. Please contact us (oligo@allelebiotech.com or 858-587-6645) for further details.

Once your order is placed, we will contact you about plasmid shipment and provide you with a time frame for completing the project.

Note: If the total project price is more than $1000, a non-refundable down payment (30% of the total service price) is registered before the project is initiated.

New Product of the Week 121310-121910:

pORB-ICAM/mWasabi-sIRES-VSVG for insect and mammalian cell expression of human membrane protein ICAM, validated and ready to ship. Email vivec@allelebiotech.com

Promotion of the Wee 120610-121310:

5% discount on high quality cell culture dishes manufactured by Phoenix Biomedical, email oligo@allelebiotech.com with code PH121310EC.

Tags: custom virus packaging, EF1a, lentivirus, lentivirus packaging, LTR, retrovirus, retrovirus titering, SV40