Protein Expression and Purification
Protein Expression and Purification
A complete line of services related to the expression of recombinant proteins using the baculovirus expression system is available. Bionexus services include production, purification and characterization of recombinant baculovirus clones in addition to scale-up for protein expression. Culture volumes from 500mL to 100L are available using either serum-containing or serum–free media formulations. Bionexus expertise in the development and optimization of serum-free medium make us an ideal choice for the conversion of existing serum-containing processes to serum-free conditions.Optimization of protein expression is accomplished using titration experiments to determine the optimal viral infection ratio and by screening various cell/media combinations. Cell separation, lysis and protein purification services are also available. We work closely with our customers to ensure that experiments are designed correctly from the initial cloning to optimization, scale-up and production. Our work is performed under strict confidentiality.
In addition, our team has significant experience in producing, isolating and purifying recombinant and natural-source proteins. Our team has produced and purified components from a variety of expression systems including procaryotic and eucaryotic expression systems such as E. coli, HeLa-Vaccinia, 293-Adenovirus, Baculovirus and CHO. Natural source proteins have been isolated from embryonic material, whole organs, tissue slices and primary cell cultures established from tissue explants.The protein isolation lab maintains equipment for the homogenization and extraction of proteins from whole organs or tissue sections, the concentration of cell culture supernatant and the lysis of whole cell suspensions. The protein purification lab supports the use of small and large volume low-pressure liquid chromatography systems for the purification of proteins by a variety of techniques. Our team members are skilled in the application of ion-exchange, size exclusion, hydrophobic interaction and affinity chromatography techniques to the purification of a wide variety of proteins. Protein purity is assessed using PAGE and Western Blot analysis as well as ELISA. Biological Activity is determined through the use of bioassays and enzyme activity assays. Rapid total protein determinations are generated by our team’s QUICK PRO protein assay kit.
The production material is further processed and purified by our team. These services may include
purification protocol development and optimization to provide maximum active material. We are
experienced with the following protein purification techniques:
- Ion-Exchange Chromatography
- Affinity Purification
- 6-His Tagged Proteins
- GST Tagged Proteins
Protein Expression Service in E.coli:
|Cloning of a gene into a bacterial expression vector|
-Amplification/isolation of the gene of interest out of a customer-supplied vector and subcloning it into a bacterial expression vector.
-Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.
- Maxi-prep of the recombinant vector DNA.
|Generation and identification of bacterial colonies expressing target proteins|
-Transformation of recombinant constructs into high efficiency expression bacterial strain.
-Mini-induction to over-express the target protein.
-Test for the expression of the recombinant protein by western blot (customer must provide appropriate antibodies) if desired.
One to two liter of bacterial culture will be induced with IPTG and harvested for protein purification.
Protein purification using Ni-NTA (for 6xHIS-tagged proteins) or glutathione (for GST-tagged proteins) beads at either native or denatured conditions.
Protein Expression Service in Baculovirus:
|Cloning of a gene of interest into a baculoviral expression vector|
|Generation, identification of baculoviral colonies expressing target proteins|
|Plaque purification of baculoviral colonies expressing target proteins|
|Amplification of recombinant baculovirus for high titer stock|
|Large-scale culture of recombinant baculovirus, per liter|
Subclone your cDNA of interest into a baculovirus transfer vector, co-transfect with baculovirus DNA, amplify resulting virus.
(Additional charge for linkers if necessary or non-standard transfer vectors. Virus may require cloning for large scale expression - see below).
Transfect and amplify transfer vector you provide
The transfer vector you provide will be co-transfected with triple-cut baculovirus DNA and the resulting virus will be amplified to100 ml of a high-titer stock Titer will be determined by the limiting dilution method. (turnaround time is 2 - 4 weeks from when your vector arrives). The virus from this procedure is usually about 95 - 99% recombinant and may require further cloning for large scale infections.
Limited dilution cloning
Client's virus will be cloned by limiting dilution on 96 well dishes. Five clones will be used to test infect one T-25 flask each and the pellet (and media if appropriate) will be shipped to you for further analysis. The chosen clone will be amplified to 250ml and titered.
Cloning by plaque purification
Client's virus will be cloned by plaque purification on agarose dishes. Five clones will be used to test infect one T-75 flask each and the pellet (and media if appropriate) will be prepared for further analysis or sent to you for analysis (shipping charge will be added).
Determine the titer of your virus by limiting dilution
Viral titer will be determined by limiting dilution on Sf-9 cells.
Single plate: Inquire
Duplicate plates: Inquire
Electrophoresis and Western blot of viral clones
Cells expressing your viral clones and appropriate controls will be harvested, solublilized in LDS sample buffer, applied to a gel, electrophoresed, blotted and probed with an antibody you provide or using any commercially available antibody. Detection by ECL. Up to 12 clones per gel. Substantial discount for multiple gels run at the same time.
Price: $980 plus cost of the first antibody
Infect and harvest 109 insect cells (1 liter culture)
We will amplify your virus if necessary and infect insect cells at high MOI and harvest 72 hours later. Determination of viral titer is included. Volume discounts for > 1L.
Electrophoresis and Western blot of subcellular protein fractions
Cells expressing your protein and appropriate controls will be fractionated into cytoplasmic, detergent-soluble and detergent-insolouble fractions, subjected to gel electrophoresis, blotted and probed with an antibody you provide or using any commercially available antibody. Detection by ECL. Up to 4 different viruses may be analyzed per gel. Substantial discount for multiple gels run at the same time.
Price: (Inquire) plus cost of the first antibody
Purify affinity-tagged protein from 1 liter of culture
Client's protein will be solubilized and purified by one cycle of affinity chromatography using the method of your choice. Includes protein gel electrophoresis of column fractions and final dialysis against PBS or other standard buffer. Non-standard buffers or other media additions may be subject to a materials charge. Substantial per liter discounts given for amounts greater than 1 liter processed at the same time.
Price: (Inquire) plus cost of affinity media
Purify protein from 1 liter or more of culture using your purification protocol
We will use a purification protocol of your design to purify your expressed protein. Cost will depend on the number of steps, cost of materials, etc.
Develop additional purification methods for your protein of interest
We will work with you to develop and implement a purification strategy for your protein
Test expression in insect larval
Infect 20 T. Ni larva, prepare an insect lysate and do PAGE and Western analysis of the protein using your antibody or a commercially available anti-6His Mab. Soluble, detergent-soluble and insoluble fractions will be analyzed.
Large scale larval protein expression and purification
Infect 100 insect larva, isolate and affinity purify your protein. (For quality assurance, must be ordered after or in addition to text expression (above)).
Price: (Inquire) plus of affinity media
Each additional 100 larva ordered concurrently, Price: (Inquire)
Sf 9 cells
A frozen vial of low passage Sf-9 cells or a T-25 flask of cells (your choice) will be provided.
All communications for this Service will be held in the strictest confidence.
Contact Bionexus for a price quotation and more information today.