protein expression

Using Insect Cells For Making Mammalian Proteins

Recombinant protein expression is a major part of biological research. In theory, once the genetic code of a protein is known from cDNA analysis or whole genome sequencing, any polypeptide of interest, existing in nature or perceived, can be artificially produced. Bacteria cells are commonly used to express a variety of proteins because they are more convenient and less costly than other systems. However, a significant percentage of proteins naturally expressed in mammalian cells are not soluble or cannot be easily produced in bacteria such as E. coli. Like bacteria, yeasts are also easy to culture and manipulate, however, although they are eukaryotes, they are not capable of adding “mammalian-like” post-translation modifications (PTM). Insect cells can be used effectively for producing large quantities of mammalian proteins rather easily through baculovirus such as Allele´s Sapphire system. PTM in insect cells is not exactly the same as in mammalian cells, e.g. different glycosylation patterns, but is a lot closer than yeasts. Mammalian cells are used for proteins that require appropriate PTM or are not soluble in other systems through either transient transfection or stable cell line establishment.

For protein expression in insect cells, a number of factors need to be taken into consideration:

1) Genomic DNA for creating baculovirus stocks that will ensure a high percentage of recombinant virus (to avoid wild-type, non-producing virus)
2) Transfer plasmid for cloning the protein-encoding cDNA for easy cloning and appropriate co-expression of helper or marker proteins (such as through insect IRES)
3) Cell lines that have the highest expression levels of a particular protein, sometimes a number of cell lines need to be screened
4) Cell medium, because insect cell medium may contain high levels of ions that can interfere with affinity tag-based purification, one needs to find the most appropriate medium for protein expression
5) Secreted vs nonsecreted proteins. Insect cells need to have their own secretion signal (and translation signal, IRES, polyadinylation, etc.)

More reading…

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Wednesday, February 29th, 2012 Viruses and cells No Comments

Commonly Known Facts About Viral Packaging -That Might Not Be Correct…

Packaging lentiviruses or retroviruses is not a routine procedure that every biology lab performs even if there is need to use it. A viral packaging protocol normally begins with preparation of purified transfer plasmid DNA, a miniprep should be enough for a few transfections. The virus backbone plasmid is either co-transfected into commonly used cells with helper plasmids that provide the essential proteins required for particle packaging, or transfected into established helper cell lines that express the required proteins from integrated transgenes. After incubation of packaging cells for a couple of days, viruses are collected and tittered. Titer determination is somewhat tricky for the inexperienced. Using a control virus expressing a fluorescent protein can make this step convenient.

Commonly known facts:

1) Lentiviruses are packaged at a titer of 10^6 IU/ml without concentrating steps.

This needs update since with more advanced technologies lentiviruses can be packaged routinely at 10^8 IU/ml. With further concentrating, the titer can be easily above 10^11 IU/ml. Retroviruses can be packaged to similar titers as well.

2) Using packaging cell lines gives the highest possible titer

While packaging cell lines (such as Allele’s popular Phoenix Eco and Ampho cells for retrovirus packaging) provides maybe the most convenient method for packaging, the yield will not reach the highest potential. Packaging cell lines may also lose their capability for packaging after continued culturing, requiring periodic selection with antibiotics and functional tests, as we do here at Allele.

3) Retroviruses are always collected in one shot after transfection into packaging cells

If the transfer vector has oriP/EBNA1 episomal maintenance system, such as some of the Phoenix vectors Allele offers, the plasmids may continue to express for up to 30 days. With puromycin selection, the titer of retrovirus produced from Eco or Ampho cells can reach 10^7 IU/ml.

This week’s promotion (102509-103109): 10% off across the board of Allele Biotech’s custom services, for an example, check out our world-leading baculovirus protein expression.

New Product/Service of the Week: Introduction of Custom Viral Packaging Service. Routine titer of 10^8 IU/ml, as high as 10^10 IU/ml, option to include cloning. Signature service ABP-CS-MERV002 provides more than 200 million particles at $7/million particles. These are game-changing prices for the viral packaging service market based on superior technologies!

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Wednesday, October 28th, 2009 Viruses and cells No Comments

Introducing Baculo Virus Expression System (BVES) with a Strong IRES

Internal ribosome entry site (IRES) can be used to initiate translation of a second open reading frame (ORF) of an mRNA, providing the benefits of: 1) avoiding promoter competition in a dual promoter situation; 2) having controlled ratio of expression of two proteins; 3) placing a dominant selection pressure on the entire bicistronic mRNA and hence the maintenance of the transgene when a selection marker is placed as the second ORF.

IRES elements are located mainly in RNA viruses except certain mammalian and insect mRNA molecules. Only one DNA virus has so far been found to contain an IRES, the while spot syndrome virus (WSSV) of marine shrimp. This IRES, compared to a very few other choices known to function in insect cells such as the IRES from Rhopalosiphum padi virus (RhPV), has strong translation initiation activity (~98-99% in reference to cap-dependent initiation), insect cell specificity, and encompasses only 180 base pairs.

Allele Biotech, with its acquisition of Orbigen, is a major provider of BVES products and services with more than 10 years of experience. Allele’s featured New Products of the Week* this week are WSSV IRES containing baculovirus vectors, the sIRES (for Strong IRES from Shrimp virus) series plasmids. Currently one version is pOrb-MCS-sIRES-VSVG for pseudotyping baculoviruses (within the Emerald Baculovirus for Mammalian Expression series), with pOrb-mWasabi-sIRES-VSVG as a fluorescent protein control; the other is pOrb-MCS-sIRES-MCS for cloning a custom second cDNA. New versions in the future will include IRES driven mWasabi and other commonly used selection markers.

With a current research project for the National Cancer Institute (NCI) within the National Institutes of Health (NIH) involving development of modified BVES and mammalian protein expression and purification systems, Allele Biotech expects this product line to continue its expansion at a fast pace.

* Allele Biotech announces at least one new product every Wednesday through news release at AlleleNews or Allele Blog and social networks.

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Wednesday, September 30th, 2009 Viruses and cells No Comments

Effective Use of Resources in Difficult Times

In scientific research, there is a tendency to have everything done in our own lab just so that you can say so, after all, scientific credit is the core criterion researchers are evaluated on. You say wait a minute, don’t we always encourage exchange of materials and COLLABORATION on projects? Sure, but not often enough to make “encouragement” unnecessary. Many “collaborations” are more like sharing of materials with conditions.

In business, collaboration is more in the form of OUTSOURCING or CO-DEVELOPMENT (sometimes through licensing), because doing everything by one’s own employees just doesn’t make much financial sense even for the mega-sized, we-have-everything type of companies. One friend of ours working at a Johnson & Johnson site once told us that a line of research using gene silencing technologies was debated but never moved forward because the lack of confidence in expertise: we are not expert on RNAi, how do we trust our own data? For most biotech and early-stage pharma companies, hiring an expert to do a task brings about too much uncertainty, not to mention cost efficiency.

“Having the expert do it” by outsourcing is somewhat more acceptable to the industry than the academia because the “We are the experts” mentality is more dominant in the latter. Heck, if we don’t believe “We are the experts” in our own field of research, then why do we even do it in the first place? In business though, who is the expert is not something one fights for if the end product or contribution to profit is not made.

The current economic conditions caused many large biotech and pharma companies to lay off thousands upon thousands of employees, in one case of Pfizer layoff, scientist positions were particularly targeted for elimination. Life goes on. Economic downturns are also opportunities for becoming lean and mean, using ways of doing things with much improved efficiency and productivity such as outsourcing, and finding new areas for long term growth.

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Monday, February 9th, 2009 State of Research No Comments