Mammalian Expression by Transient Transfection
Subcloning/PCR Cloning
BPS will clone your insert into the desired mammalian expression vector. Either standard subcloning or PCR cloning will be used to transfer the gene of interest into the expression vector. Clones will then be screened for the correct restriction fragments and sequenced.
Optimization of Expression and Protein Characterization
The mammalian expression vector will be transiently transfected into suspension 293 cells grown in a defined, serum-free medium. Recombinant protein production will be optimized in a small-scale culture by doing a time course of expression. For each test, the recombinant protein will be analyzed by SDS-PAGE and/or Western blot. In addition, the yield per unit volume will be estimated. Using the results of these experiments, a strategy for producing and purifying the protein will be made.
Protein Production
Production of the recombinant mammalian-expressed protein can be scaled up to 1 liter. You will receive the cell pellets or conditioned media and a complete project report with the SDS-PAGE/Western blot analysis that documents the protein production.
Protein purification
BPS will purify your recombinant proteins using an affinity column. Large scale purification is also available.
Mammalian Stable Cell Line Development
BPS provides a custom service to generate stable cell lines that constantly express proteins of interest at high, homogeneous levels. The gene of interest will be cloned into the desired mammalian expression vector. The parent cell line will then be tested for its sensitivity to selection antibiotics and optimized for transfection conditions. The stable cell line is generated by the transfection of the expression construct into the parent cell line with subsequent selection with antibiotics. The individual stable clones will be expanded and screened by Western Blot for optimal expression of the protein of interest. Quality control testing will be performed to ensure that the final stable cell lines meet the requirements of the customers.
If you would like to learn more about our mammalian expression service, please contact us.
Subcloning/PCR Cloning
BPS will clone your insert into a baculovirus transfer vector. Either a standard subcloning or a PCR cloning will be used to transfer the gene of interest into the vector. Clones will be screened for the correct restriction fragments and sequenced, if necessary.
Recombinant Virus Production
Using linearized DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV), BPS will cotransfect a baculovirus transfer vector into Sf9 cells. The low –titer recombinant viruses will be amplified 2-3 times to generate high-titer virus stocks.
Optimization of Expression
Recombinant protein expression will be optimized in one or more cell lines by doing a time-course study of infection, varying the multiplicity of infection (MOI). At each time point, the expressed protein will be analyzed by SDS-PAGE and/or Western blot. The results of these experiments will be used to determine the optimal conditions for protein production.
Protein Characterization
Recombinant protein from a small-scale culture will be characterized for cellular location, solubility, and accessibility of purification tags. In addition, the yield per unit volume will be estimated. Using the results of these experiments, a strategy for producing and purifying the protein will be made.
Protein Production
Production of the recombinant protein can be scaled up between one and several liter volumes. You will receive the cell pellets or conditioned media and a complete project report with the SDS-PAGE and/or Western blot analysis that document protein production.
Protein Purification
BPS will purify your recombinant proteins using an affinity column. Large scale purification is also available.
If you would like to learn more about our baculovirus expression service, please contact us.
Subcloning/PCR Cloning
BPS will clone your insert into an expression vector. Either a standard subcloning or a PCR cloning will be used to transfer the gene of interest into the expression vector. Clones will be screened for the correct restriction fragments and sequenced.
Expression Screening
The expression vector containing the gene of interest will be transformed into an expression strain of E. coli for protein production. Three to five individual clones will be screened in a small-scale induction experiment and analyzed by SDS/PAGE for production of a protein of the expected size. If desired, a Western blot can be carried out to verify immunoreactivity with your antisera, or with an anti-affinity tag antisera.
Optimization of Expression
The expression of the recombinant protein can be optimized by doing a time course of induction, altering induction temperature or screening growth media. For each test, the recombinant protein will be analyzed by SDS-PAGE and/or Western blot to determine optimal conditions for protein production.
Protein Characterization
A small-scale culture (10-100 ml) from one isolate of the strain producing the recombinant protein will be induced and the expressed protein will be characterized for cellular location, solubility, and accessibility of purification tags. In addition, the yield per unit volume will be estimated. Using the results of these experiments, a strategy for producing and purifying the protein will be made.
Protein Production
Using shake flasks the strain expressing the recombinant protein can be scaled up between one and several liter volumes. You will receive the cell pellets and a complete project report with the SDS-PAGE and/or Western blot analysis that document protein production.
Protein Purification
BPS will purify your recombinant protein(s). We offer a variety of services from inclusion body preparation to affinity column purification.
If you would like to learn more about our E.coli expression service, please contact us.
BPS provides mAb transient expression service by using serum free HEK293 suspension system.
Pilot expression and purification will be evaluated and optimized. Using the results of these experiments, mAb expression will be scaled up and purified by affinity chromatography. You will receive the purified antibody and a complete project report with the SDS-PAGE/Western blot analysis that documents the antibody production.
Case Study
Figure: SDS-PAGE analysis of human Antibody A transiently expressed in our 293 expression system.
293 cells grown in serum free medium were transiently transfected with expression vector for Antibody A. Three days after transfection, conditional medium were harvested and incubated with Protein A agarose beads. The antibody bound protein A agarose beads were spun down and washed with PBS, then SDS protein sample buffer with or without DTT was added to the beads and boiled. Samples were loaded onto 4-20% gradient SDSPAGE and stained with coomassie blue.
Lanes 1 and 2: Antibody A under non-reducing condition. The input conditional medium was ~0.5 ml for lane 1 and ~0.25 ml for lane 2.
Lanes 3 and 4: Antibody A under reducing condition. The input conditional medium was ~0.5 ml for lane 3 and ~0.25 ml for lane 4.
Lanes 5-7: purified BSA standard. The amount of BSA is 5 μg for lane 5, 2 μg for lane 6 and 1 μg for lane 7.
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