List of Services

Genetic Engineering and Protein Expression

Service Description/Comments
Plasmid Preparation Plasmids are amplified in appropriate E. coli hosts, most commonly DH5aF', and analyzed by agarose gel electrophoresis as intact and restriction digested DNA, unless customer specifies otherwise. Plasmids can be provided by customer, purchased by ARVYS on behalf of customer or used under license to ARVYS.
DNA amplification DNA is amplified by high fidelity PCR using primers designed at ARVYS or specified by the customer. Primer synthesis is outsourced. The amplified fragment is restriction digested and purified.
Clone generation Amplified DNA of interest is inserted into an appropriate plasmid and transformed in E. coli. Clones are generated by antibiotic resistance or media selection and 2 rounds of single colony purification.
Colony PCR-screening Single colonies are screened by PCR to identify the correct clone.
Restriction mapping This service is carried out primarily to ensure the correct structure of a vector in areas that are not sequenced.
Consensus sequence derivation One analysis of alignment to reference is performed.
DNA sequencing DNA sequencing is outsourced at cost. To ensure value of this service ARVYS works with high quality providers proficient in determining simple and difficult (e.g., GC-rich) sequences.
DNA synthesis DNA synthesis is outsourced at cost.
Expression in E. coli Expression in E. coli encompasses design of an optimal insert/vector/host combination, vector and fragment preparation, ligation, transformation, colony purification, colony PCR, DNA sequencing, plasmid mini-prep and storage, transformation into expression host and expression analysis by SDS-PAGE/Protein stain.
Expression vector optimization Includes experimental design, modification of the vector, promoter, ribosome binding site, or gene, ligation, transformation, colony purification, colony PCR, DNA sequencing, plasmid mini-prep and storage, expression analysis by SDS-PAGE/Protein stain.
E. coli strain selection E. coli strain selection encompasses vector transformation into various hosts, colony selection, stock culture preparation and expression analysis by SDS-PAGE/Protein stain.
E. coli culture optimization Effects of media, temperature and induction conditions on protein expression are determined. Level of expression and product distribution between soluble and insoluble fractions are analyzed by SDS-PAGE.
Expression analysis for an existing E. coli system Bacteria carrying the gene of interest are grown and expression is induced, if necessary. Growth is monitored by optical density. Cultures are sampled at three time points and protein expression analyzed by SDS-PAGE. Distribution of expressed protein between soluble and insoluble fractions is analyzed upon request.
Transient expression in mammalian cells Engineered vectors are transfected in appropriate cell line and selected by antibiotic or other resistance. Cell growth and viability are monitored, and protein expression analyzed by SDS-PAGE/Western/dot blot/ELISA.
Stable expression in mammalian cells Engineered vectors are transfected in appropriate cell line and selected by antibiotic or other resistance. Stable transfectants are monitored for growth and viability and amplified. High producers are identified by SDS-PAGE/Western/dot-blot/ELISA, selected by limited dilution and confirmed by SDS-PAGE/Western/dot blot/ELISA and PCR.
Service Description/Comments
Cell line initiation The cell line is grown from a frozen vial to a single T175 flask.
Conditioned media production Cells are grown in triple flasks or roller bottles. Viability and growth data are generated from a parallel representative culture.
Mammalian cell mass production from adherent cultures Cells are grown in triple flasks, roller bottles, or NUNC Factory. Viability and growth curves are generated from a parallel representative culture.
Mammalian cell mass production from suspenstion cultures Cells are grown in triple flasks or roller bottles. Viability and growth curves are generated from a parallel representative culture.
Protein expression monitoring Protein expression samples are collected at 7 time points from production culture and analyzed by SDS-PAGE/Western or ELISA. Other assays can be performed upon request.
Bacterial cell paste production Cells are grown to a specified density and induced, when necessary. Protein expression samples are collected at 3 growth points and analyzed by SDS-PAGE/Coomassie.
Service Description/Comments
SDS-PAGE/Protein stain Samples are run on 10- or 15-well premade gels. The gels are stained, scanned and documented.
Native or Urea PAGE Samples are run on 10- or 15-well premade gels. The gels are stained, scanned and documented.
IEF-PAGE/Coomassie Samples are run on 10-well NOVEX premade gels. The gels are stained, scanned and documented.
Zymography Zymography is performed for qualitative evaluation of proteolytic activity.
SDS-PAGE/Western Samples are run on 10- or 15-well premade gels and transferred to nitrocellulose or PVDF membrane. Blots are probed with target protein-specific reagents and detected by chemiluminescence. Films are scanned and documented.
Dot Blot Samples are dotted onto nitrocellulose membrane in 2µl aliquots or using a 96-well dot blotter. Blots are probed with target-specific protein reagents. Films are scanned and documented.
Dot/Western blot optimization Dilutions of the primary, detection and isotype control antibodies and blocking conditions are optimized. Upon request further studies can be conducted. These studies might include characterization of antigen-antibody interaction in terms of its sensitivity to pH, presence of detergents, denaturation, heat, deglycosylation, oxidation, etc.; optimization of blotting and transfer conditions.
Antibody isotyping Antibody isotype and the presence of contaminating antibodies of other isotypes are determined in a strip format.
Protein assay BCA or equivalent protein assay is performed in a 96-well plate format. Up to 5 samples per plate can be analyzed.
Light absorbance at 280 nm
Contaminating DNA assay Fluoresence-based assay used for analysis of DNA impurities in protein preparations. Up to 5 samples per plate can be analyzed.
UV-Vis absorption spectrum
Purity analysis To analyze protein purity, the densitometry of a Coomassie-stained gel is performed. Percent purity is calculated in the linear range of target and contaminating proteins. Samples can also be prepared and submitted for N-terminal sequencing and mass-spectroscopy analysis.
Endotoxin concentration Kinetic LAL assay is performed for determination of endotoxin concentration. Up to 5 samples per plate can be analyzed.
Protein deglycosylation analysis O-linked and N-linked glycochains are sequentially removed from glycoproteins, and the samples are analyzed by SDS-PAGE for molecular weight determination.
Enzyme kinetics Enzyme kinetic parameters (Km, Vmax, Ki, IC50) are determined.
Ligand binding Given the availability of a fluorescence-based assay, Kd, Bmax and Hill coefficient, kinetics of ligand association and dissociation are determined.
Protein-protein interactions Protein-protein interactions are characterized by coimmunoprecipitation, chromatography, spectroscopy-based assays or BIACORE.
Service Description/Comments
Bacterial paste or tissues/cell pellet lysate preparation Cells/tissues are disrupted mechanically or with a detergent, centrifuged and the soluble and insoluble fractions are collected.
Protein extraction from insoluble pellet/membranes Proteins are extracted from insoluble material using customer supplied protocol, or protocol selected by ARVYS. We offer various extraction options to suite most target proteins. If needed, an extraction step can be developed by testing different extraction conditions on a small scale.
Extraction and recovery of inclusion bodies from E.coli pellet. Bacterial pellets are washed and inclusion bodies are extracted with quanidinium, urea or detergents.
Refolding screen 10 refolding conditions are tested. Success of refolding is analyzed by solubility of the target protein.
Refolding of solubilized inclusion bodies Solubilized inclusion bodies are refolded by dialysis under a specified set of refolding conditions, such as protein concentration, rate of refolding and excipients.
Fractionation by ammonium sulfate precipitation This service can be performed by following existing protocol or by a method developed by ARVYS. For method development, 8 concentration points are tested on small sample aliquots. Precipitation fractions are analyzed by SDS-PAGE/Protein stain, Western blot or an activity assay.
Partition into Triton X-114 This method is very useful for fractionation of integral membrane proteins or as an additional step for endotoxin removal.
Sample concentration Samples are concentrated by tangential flow (TFF) or centrifugal filtration.
IgM fractionation IgM antibodies are fractionated from conditioned media or ascites fluid to ~80% purity in a single precipitation step. This fraction is a starting material for further IgM purification.
Chromatography Chromatography steps follow customer-supplied protocols, published protocols, standard protocols (for antibodies or tagged/fused proteins only) or protocols developed by ARVYS. Our standard chromatography modes include protein A/G, gel filtration, ion exchange, tag affinity, hydrophobic interaction. Dye affinity, nucleotide affinity, hydroxylapatite and other chromatography modes can be used if needed. Each run is performed on a dedicated column. We use HiPrep premade columns from Amersham Pharmacia for most purifications and custom-made columns for large scale purifications. Runs are monitored by light absorbance at 280 nm and conductivity. Fractions are analyzed by SDS-PAGE/Protein stain or dot blot analyses, other assays can be performed when necessary.
Development of affinity media Ligands are immobilized on a chromatography matrix according to customer-specified or ARVYS selected conjugation method.
Endotoxin removal The endotoxin removal protocol are designed to meet customer-specified endotoxin levels. Endotoxin concentrations are measured by LAL kinetic assay.
Tag/Fusion protein removal Tags or fusion proteins are digested from target proteins according to standard protocols. Extent of digestion, removal of tag/fusion protein and of cleaving enzyme are analyzed by SDS-PAGE/Western blot and SDS-PAGE/Protein stain. If required, product-specific optimization of the digestion protocol is performed.
Preformulation screening Following evaluation of the protein's sensitivity to pH, heat and oxidation various buffer conditions are tested. Buffers that provide maximal protein stability and minimal protein degradation are selected.
Lyophilization
Final sample preparation The purified protein sample is analyzed by SDS-PAGE/Coomassie. Protein concentration of the solution samples is determined from known or theoretical extinction coefficients at 280 nm, or weight measurement for a lyophilized product. Additional assays are available upon request. Final samples are prepared according to customer specifications.
Service Description/Comments
Dot/Western blot optimization Optimization of detection - dilutions of the primary, detection and isotype control antibodies, blocking conditions are optimized.

As a separate service determination of antibody-antigen interaction sensitivity to the buffer composition, heat and pH, optimization of blotting and transfer conditions is available.
Direct ELISA Up to 5 samples per plate can be analyzed. Direct ELISA method development includes selection of a detection antibody, optimization of binding and detection conditions, determination of assay sensitivity, linearity range, recoveries of known amounts of standard preparations and intra-assay coefficient of variation (CV).
Sandwich ELISA Up to 5 samples per plate can be analyzed. Sandwich ELISA method development includes selection of an antibody sandwich pair, optimization of binding and detection conditions, determination of assay sensitivity, linearity range, recoveries of known amounts of standard preparations and intra-assay coefficient of variation (CV).
Enzyme activity assay Enzyme activity assays are performed according to customer-supplied protocols, published protocols or protocols developed by ARVYS. Preferred detection methods include light absorbance, fluorescence intensity or polarization, fluorescence resonance energy transfer and luminescence.
Ligand binding assay Given availability of a fluorescent probe, the receptor-ligand assays are performed according to customer-supplied protocols, published protocols or protocols developed by ARVYS.
BIACORE analysis Please inquire

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