ARVYS Proteins Inc. is a Contract Research Organization (CRO) that Specializes in Custom Protein Services for Drug Discovery and Life Science Research. Our strong integrated expertise in protein biochemistry, working experience with various recombinant expression systems and protein classes, up-to-date knowledge in protein technologies, enable us to become a reliable and resourceful partner for our clients. We help at any stage of a protein project. Our goal is to become your preferred outsourcing choice for Protein Services and we will work hard to earn your business.

OUR CUSTOM PROTEIN SERVICES

Protein Expression

Protein Expression

Generation of heterologous recombinant proteins in multiple expression systems
Fermentation & Cell Culture

Fermentation &
Cell Culture

Bacterial, yeast, insect and mammalian biomass production for scale up processes
Protein Purification

Protein Purification

Homogeneous and well-characterized preparations save time, effort and resources
Protein Characterization

Protein Characterization

Customized design to address specific protein characterization goals
Functional Assays and Assay Development

Functional Assays & Assay Development

Protocols with detection by UV-Vis, fluorescence or luminescence spectroscopy
ELISA Developmentn

ELISA Development

From selection of assay components to development of a working protocol
Protein Labeling and Conjugation

Protein Labeling & Conjugation

Development of strategies to maximize functionality of modified proteins
Endotoxin Removal and Testing

Endotoxin Removal & Testing

Expert service in less than 1 week
Antibody Development & Production

Antibody Development & Production

From generation of antigen to characterization of antibodies

ARVYS Proteins Inc. offers more

Looking for additional services? We offer even more services than listed, contact us to find out how we can assist you. Using latest advances in Protein Technologies ARVYS Proteins Inc. strives to provide the highest quality custom protein services in the industry. We work closely with you, analyze your project at the planning stage and take several approaches to ensure you get the results you are looking for.
Services

Protein Expression

Our Protein Expression Services incorporate the latest developments in bacterial, baculovirus and mammalian expression technologies to assure that your recombinant protein production will be efficient and economical. We offer a number of Protein Expression Services designed to meet specific goals, ranging from expression of tagged proteins for research to production of biopharmaceuticals for clinical use. Your custom protein expression will be optimized by engineering an expression construct with a codon-optimized DNA insert, a strong promoter, an efficient ribosome binding site, a high copy number and combining it with an appropriate host. High level of protein expression also depends on stability, solubility, and folding pathway of the protein product. Accordingly, we will optimize these protein expression parameters when such optimization is necessary.

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GENETIC ENGINEERING

  • generation of a gene by PCR or synthesis
  • subcloning into an appropriate vector
  • confirmation of DNA insert by Sanger sequencing
  • plasmid preparation and DNA storage
  • site-directed mutagenesis

BACTERIAL PROTEIN EXPRESSION

  • codon optimization for bacterial expression and gene synthesis of DNA insert
  • mutagenesis of an existing construct
  • generation of bacterial expression construct(s) in a selected or client-specified vector
  • transformation into an appropriate host and preparation of glycerol stocks
  • screening studies for the best-expressing protein variant 
  • optimization of growth conditions (host, induction, media, temperature, additives) to drive either soluble or inclusion bodies expression 
  • soluble vs. inclusion bodies expression assessment by SDS-PAGE/Coomassie or Western blot
  • bacterial paste scale up production (read more Fermentation & Cell Culture)
  • inclusion bodies wash and recovery
  • recombinant protein purification from soluble lysate fraction (read more Protein Purification)
  • recombinant protein purification from inclusion bodies by refolding (read more Protein Purification)

BACULOVIRUS-INDUCED INSECT PROTEIN EXPRESSION

  • codon optimization for insect cell expression and gene synthesis of DNA insert
  • mutagenesis of an existing construct
  • subcloning of a DNA insert into a selected or client-specified baculovirus expression vector 
  • virus generation, amplification and cloning by limited dilution or plaque purification 
  • generation of high-titer virus stocks
  • SDS-PAGE, Western, ELISA or functional expression analysis of cell lysates and conditioned media for intracellular and secreted protein products respectively
  • secreted or intracellular expression in Sf9, Sf21 and HiFive insect cells
  • expression optimization studies: MOI, time course, growth media
  • large-scale insect cell culture for conditioned media or cell pellet production  (read more Fermentation & Cell Culture)
  • recombinant protein purification from conditioned media or cell pellet (read more Protein Purification)

YEAST PROTEIN EXPRESSION

  • codon optimization for yeast cell expression and gene synthesis of DNA insert
  • subcloning of a DNA insert into a selected selected yeast expression vector 
  • mutagenesis of an existing construct
  • generation of yeast transformants
  • screening for high-yielding transformants by Western, ELISA or functional expression analysis of cell lysates
  • intracellular expression of membrane proteins in S.cerevisiae yeast cells
  • secreted expression in Pichia yeast cells
  • expression optimization studies: secretion signal, host strain, media
  • large-scale yeast cell culture for conditioned media or cell pellet production recombinant protein purification from conditioned media or cell pellet (read more Fermentation & Cell Culture)
  • recombinant protein purification from conditioned media or cell pellet (read more Protein Purification)

MAMMALIAN PROTEIN EXPRESSION

  • codon optimization for mammalian cell expression and gene synthesis of DNA insert 
  • mutagenesis of an existing construct
  • generation of mammalian protein expression construct(s) in a selected or client-specified vector
  • endotoxin-free plasmid DNA preparation for transient or stable transfections
  • small-scale trial to assess recombinant protein expression and/or to optimize transient transfection conditions
  • various transient transfection reagent options
  • generation of stably-transfected cell pool
  • expression analysis by SDS-PAGE, Western, ELISA or functional assay
  • secreted or intracellular expression in CHO, HEK293, HEK293E or client-specified cells 
  • adherent or suspension cell growth options
  • large-scale mammalian cell culture for conditioned media or cell pellet production  (read more Fermentation & Cell Culture)
  • recombinant protein purification from conditioned media or cell pellet (read more Protein Purification)

STABLE CELL LINE DEVELOPMENT

AUXILLARY PROTEIN EXPRESSION SERVICES INCLUDE

  • long-term storage for DNA, glycerol and virus stocks
  • master and working cell banking

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Fermentation & Cell Culture

The quality of starting materials resulting from Fermentation Scale Up often determine the purification strategy or even the success of purification. Our Cell Culture Services aim at achieving maximal r-protein production through optimization of fermentation processes and it could be a very important purification step in itself. Time and effort invested at this stage often result in substantially shorter purification protocols and higher yields of active r-protein. Our experience with various high-producing cell lines, cell culture growth modes, media formulations, methods of protein extraction, fractionation and enrichment enables us to design the most optimal production process for your starting materials.

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LARGE SCALE PLASMID DNA PREPARATION

LARGE SCALE PROTEIN PRODUCTION

  • clarified conditioned media from mammalian, yeast and insect cells
  • mammalian cell pellet
  • insect cell paste
  • bacterial paste
  • yeast paste
  • transient transfection into mammalian cells
  • infection of insect cells with high-titer baculovirus
  • propagation of stably-expressing insect or mammalian cells
  • suspension cell line growth in shake flasks and Wave bioreactors
  • adherent cell line growth in multifloor flasks and cell factories

CELL LYSIS OPTIONS 

  • sonication
  • homogenization
  • detergent solubilization
  • hypotonic solution treatment
  • combination of the above

INCLUSION BODIES REFOLDING SCREEN

TARGET PROTEIN ENRICHMENT BY

  • ammonium sulfate precipitation
  • sucrose gradient centrifugation
  • differential detergent extraction
  • partition into Triton X-114
  • treatment for membrane preparations
  • inclusion bodies isolation, wash, solubilization and refolding

CONCENTRATION AND BUFFER EXCHANGE OF CONDITIONED MEDIA BY

  • centrifugation
  • tangential flow filtration (TFF)

QUANTITATIVE ANALYSIS OF THE TARGET PROTEIN EXPRESSION


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Protein Purification

Our skills in custom protein purification were gained over many years of work with protein pharmaceuticals, drug targets, biological system components and biochemical reagents. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization. Our purification strategy aims to achieve a homogeneous active protein preparation in two to three purification steps. This goal is reached by a thorough selection and optimization of the capture step, incorporation of a gel filtration step to remove aggregates, degradation products and other contaminants, selection of buffer conditions that stabilize biological activity and prevent product degradation. We routinely work with antibodies, antigens, enzymes, growth factors, DNA-binding proteins, membrane proteins, blood proteins and many more. Although most proteins require an individual approach, we are confident that we can handle your protein. We will purify it cost-efficiently, characterize it according to your specifications and deliver it to you in an active and application-compatible form.

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CUSTOM PROTEIN PURIFICATION FEATURES

  • purification of 
    • fusion and tagged proteins from recombinant sources
    • native proteins from natural sources
    • antibodies of different isotypes including IgMs
    • membrane proteins
  • common protein purification scales range from 0.001g to 5g
  • proteins are purified according to client-specified purity
  • protein purification methods from
    • client-supplied protocols
    • published protocols
    • improved protocols from client-supplied or published protocols
    • de novo protocols tailored to client's requirements
  • any protein purification mode can be used
    • ion-exchange
    • gel filtration
    • affinity (broad-spectrum)
    • hydrophobic interaction 
  • refolding from inclusion bodies 
  • protein purification method development for transfer to a GMP facility
  • dedicated columns are used in each project
  • efficiency is provided by the automation and precision of AKTA systems from Amersham BioSciences (currently GE)

ANALYSIS OF INTERMEDIATE AND FINAL PURIFIED PROTEINS

SDS-PAGE and/or dot/Western blotting are routinely used for fraction analysis final protein purity is determined by densitometry from Coomassie-stained gels final products are supplied with a certificate of analysis, purification report and, if applicable, a batch record

  • SDS-PAGE and/or dot/Western blotting are routinely used for fraction analysis
  • final protein purity is determined by densitometry from Coomassie-stained gels
  • final products are supplied with a certificate of analysis, purification report and, if applicable, a batch record

ALL CUSTOM PURIFIED PROTEINS ARE

  • supplied with a certificate of analysis tailored to client's specification (read more Protein Characterization)
  • provided at specified protein concentration
  • formulated in a buffer that protects from protein degradation due to proteolysis, oxidation and shear stress
  • dispensed into specified aliquot sizes

AUXILIARY PROTEIN PURIFICATION SERVICES INCLUDE


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Protein Characterization

Analytical characterization ensures the identity, purity, structural and conformational integrity, and function of the protein. We perform a number of routine protein analyses throughout all project stages. If needed, we can submit your samples for additional methods of characterization. We also offer specialized protein characterization services for purified proteins.

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ANALYSIS OF RAW, INTERMEDIATE AND FINAL PROTEIN PRODUCTS

  • electrophoresis (SDS-PAGE, native-PAGE, IEF-PAGE, urea-PAGE)
  • Western blot/dot blot
  • enzyme activity assay by light absorbance or fluorescence
  • ELISA (direct or sandwich)-ELISA Development
  • protein assay (A280, BCA or equivalent)
  • antibody isotyping
  • endotoxin measurement -Endotoxin Assay Quote Request
  • contaminating DNA assay
  • UV-Vis absorption spectrum

PREPARATION AND SUBMISSION OF PROTEIN SAMPLES FOR

  • amino acid analysis
  • N-terminal analysis
  • mass-spectrometry identification and analysis
  • extinction coefficient determination

PROTEIN CHARACTERIZATION

  • analytical size exclusion chromatography (native MW estimate, aggregation analysis)
  • analytical ion-exchange chromatography (oxidation)
  • purity analysis by densitometry
  • UV-Vis light absorption spectroscopy
  • analysis of oxidation, degradation and aggregation products
  • protein deglycosylation analysis
  • binding interactions by co-immunoprecipitation, spectroscopy or chromatography

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Functional Assays & Assay Development

We offer our experience in Functional Assays & Assay Development for detailed mechanistic studies of a target protein, bioanalytical measurments and high-throughput screening. Since we thoroughly characterize the materials for which an assay is developed, our assays have maximized signal windows, low variability and are economical. These assays can have diverse applications such as analysis of enzyme activities, small molecule binding to proteins, protein-protein interactions and nucleic acid-protein interactions. In addition, we offer our services for outsourcing your assays or mechanistic studies.

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FUNCTIONAL MODES

  • protein-protein, protein-nucleic acid, protein-small molecule or peptide binding interactions
  • receptor functional assays
  • enzymatic activity
  • direct, indirect, sandwich, and competitive ELISA - ELISA Development
  • inhibition

ASSAY FORMATS

  • 96-well plate
  • single sample (for UV/Vis absorbance)
  • HTS-compatible

PREFERRED DETECTION METHODS

  • fluorescence intensity
  • fluorescence polarization
  • fluorescence energy resonance transfer (FRET)
  • luminescence
  • light absorbance

ASSAY TARGETS

  • enzymes
  • antibodies
  • receptors
  • inhibitors
  • peptides
  • small molecules

ASSAY DELIVERABLES

  • binding site characterization (Bmax and Jd)
  • enzyme steady state kinetic parameters determination (Km and Vmax)
  • inhibitor characterization
  • single point inhibition for multiple inhibitors at a specified concentration
  • IC50 and Ki determination
  • analysis of tight binding inhibitor kinetics
  • concentration determination for proteins, peptides and small molecules

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ELISA Development

Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used assay in diagnostics, drug discovery, clinical development and other life-science industries. This is a sensitive assay that detects and quantifies a target molecule in biological fluids or solutions. ELISA can be performed in various formats and platforms. Although a target-specific antibody is the central aspect of an ELISA, other components are also critical for the assay performance. Our seamless, integrated expertise in antibody-antigen biochemistry, functional assays, recombinant protein production, hybridoma cell culture, antibody purification, characterization and conjugation allows us to deliver a fully optimized and validated ELISA for its intended final application.

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ELISA TYPES

  • direct
  • indirect
  • sandwich
  • competition
  • inhibition

ELISA DETECTION OPTIONS

  • visible absorbance
  • fluorescence
  • luminescence

ELISA DESIGN INCLUDES SELECTION OF:

  • assay format
  • antibody or antibody pair
  • standards
  • plate-coating chemistry
  • blocking and wash buffers
  • detection strategy
  • detection reagents

ELISA OPTIMIZATION INVOLVES TESTING OF VARIOUS:

  • concentrations of antibodies, samples and buffers
  • coating protocols
  • detection protocols
  • chessboard titrations

ELISA VALIDATION PARAMETERS ARE TESTED:

  • robustness / precision / trueness
  • uncertainty
  • limit of quantification
  • dilutional linearity
  • parallelism
  • recovery
  • selectivity
  • sample stability

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Protein Labeling & Conjugation

Labeled proteins allow us to study specific molecular interactions with high sensitivity in complex biological systems. They are important reagents in numerous biological applications such as assays, purifications, protein arrays, localization studies, flow cytometry, clinical imaging and much more. The quality of labeled proteins is critical for consistent and reliable data. Although labeling procedures appear to be simple and straightforward, most of them still need to be adjusted to take into consideration the nature of a protein in order to achieve the desired results. Possible problems during labeling procedures include protein losses due to precipitation, sample manipulation & instability, inconsistent label-to-protein ratio, incomplete removal of an unconjugated labeling probe, and poor protein characterization before and after labeling. In addition, new labeling technologies had emerged for site-directed labeling requiring an integration of protein expression and purification into the labeling process. We have extensive experience with various protein labeling techniques and are confident that we can provide you with high-quality labeled reagents for your downstream applications.

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NONSELECTIVE PROTEIN LABELING

  • biotinylation with incorporation ratios determined by a HABA-based assay
  • fluorescent probe conjugation with incorporation ratios determined by fluorescence 
  • other chemical moieties (for example, Sulfo-Tag, Dyes) with incorporation ratios determined by UV-Vis spectroscopy
  • enzyme conjugation with incorporation ratios determined by enzyme activity assays

SITE-SPECIFIC LABELING

  • C-terminal labeling through "Sortagging"
  • N-terminal labeling through "Sortagging"
  • labeling at glycosylation sites

CLICK CHEMISTRY LABELING

  • azido modified proteins and/or glycochains 
  • alkyne modified proteins and/or glycochains
  • copper (I)-catalyzed click labeling 
  • copper-free click labeling
  • various commercial choices of click partners including dual labels
  • in situ applications
  • protein conjugation and detection

BIOMOLECULE IMMOBILIZATION ON SOLID SUPPORT

  • site-specific immobilization on a chip in oriented fashion 
  • protein conjugation to chromatography resin

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Endotoxin Removal & Testing

Endotoxin Removal from biological solutions is critical for many in vivo and cell-based applications, as it interferes with biological response. Endotoxins are liposaccharides that are found in the outer cell wall of Gramm-negative bacteria. Bacteria release endotoxins at the time of lysis. The toxic effect of endotoxins is triggered by its interaction with specific receptors on the immune cells resulting in the release of high concentrations of cytokines and other molecules of immunological significance. Since adventitious endotoxin is present in air, water, labware and it cannot be removed by simple sterilization, it is almost impossible to generate endotoxin-free solutions without a removal procedure. Each Endotoxin Removal project requires a protocol development step that takes into consideration biophysical properties of a target molecule, final sample application and desired formulation. For years we had successfully developed Endotoxin Removal protocols to meet strict endotoxin presence requirements. We are confident that we can make your Endotoxin Removal project a success.

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CUSTOM ENDOTOXIN REMOVAL FROM AQUEOUS SOLUTIONS OF:

  • proteins
  • DNA 
  • peptides
  • biomass

METHODS ARE BASED ON:

  • charge
  • hydrophobicity
  • combinantion of charge and hydrophobicity
  • ligand affinity
  • size

SERVICE FEATURES:

  • protein losses are minimized by selection of an appropriate endotoxin removal method and its optimization
  • scales range from mg to g of a target molecule
  • endotoxin-free samples are sterilized and distributed into multiple aliquots for convenience
  • delivered in an application-compatible buffer
  • turnaround times are less than 1 week in most cases - Endotoxin Assay Quote Request
  • detergent-compatible r-Factor C fluorescence-based assay
  • detection levels below 0.01EU/ml
  • LAL kinetic assay and other endotoxin assays are available

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Antibody Development & Production

In the last 30 years, Antibodies had become very important reagents in research, diagnostics, and medicine. This class of molecules are heavily used now in many fields of biomedical research such as immunoassays (Western blots, ELISA, etc.), protein purification, protein identification and protein-protein interaction studies. Many commercial diagnostic kits have antibodies as their critical components. Currently, there are 75 Monoclonal Antibodies or their derivatives approved by the FDA and many more are being tested in clinical studies. Each antibody project requires a customized approach gathering all facts about an antigen and defining the properties of the final antibody. We have a long history in developing and producing antibodies for our clients. We will plan your Antibody project thoroughly to achieve desired outcomes.

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PURIFICATION OF ANTIBODIES FROM BIOLOGICAL SOURCES

  • all antibody isotypes from any species
  • endotoxin removal from antibody solutions
  • antibody conjugation to fluorescent dyes or enzymes
  • antibody characterization by ELISA and other assays

PRODUCTION OF RECOMBINANT ANTIBODIES IN MAMMALIAN EXPRESSION SYSTEMS BY TRANSIENT OR STABLE TRANSFECTION


PRODUCTION OF MONOCLONAL ANTIBODIES FROM HYBRIDOMA CELL LINES


PRODUCTION OF POLYCLONAL ANTIBODIES

  • design of an antigen with immunogenic properties
  • antigen generation, production and purification
  • analysis of bleeds for specificity by ELISA and/or Western blot
  • purification of antibodies from blood plasma

PRODUCTION OF SINGLE CHAIN ANTIBODIES (scFv) and Fab FRAGMENTS IN E.COLI

  • design of an expression construct
  • expression in E.coli and its optimization
  • antibody purification

 GENERATION OF Fab AND (Fab)2 FRAGMENTS FROM FULL LENGTH ANTIBODIES

  • optimization of enzymatic digestion reaction
  • removal of heavy chain fragments
  • Fab and (Fab)2 purification

PHAGE DISPLAY FOR GENERATION OF MONOCLONAL ANTIBODIES

  • generation of antibody phage display library from infected/diseased patients or antigen-immunized animals
  • phage display panning and screening
  • sequence analysis
  • generation of scFv, Fabs or whole immunoglobulins in various expression

 CHARACTERIZATION OF ANTIBODIES FOR SPECIFICITY AND SELECTIVITY


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