At GVK BIO, we are adept at cloning the gene of interest into choice of expression vector, right from cDNA synthesis. We have extensive experience in site directed mutagenesis/truncated/fusion protein construction.
We routinely use RT-PCR for cDNA synthesis from isolated RNA and also construct cDNA libraries. The cDNA is cloned into expression vectors and the confirmed clone is sequenced using primers for every 250-300 base pairs. We employ various strategies like Gateway, TOPO, Tet-on/off, Fusion and Tag Constructs, based on the client’s requirement.
We also perform mini to giga scale preparation for DNA isolation. The isolated DNA is subject to several quality control checks, agarose gel electrophoresis, restriction digestion, endotoxin testing and DNA sequencing using primers for every 250-300 base pairs.
With a processing capacity of 5-50 litre culture volume, we offer extensive expertise across all the purification techniques using AKTA Explorer platform. The steps employed in purifying the proteins mainly focus on purity, yield and recovery. Complete knowledge and hands-on experience at each step of purification enables us to select an apt technique for the required protein purity and yields to generate milligram to gram level of reagent.
- E.coli – (Bl21(DE3), BL21(DE3)pLysS, BL21, Rosetta.2(DE3), C41 and C43)
- Insect cell – (Sf9, Sf21)
- Mammalian – CHO and HEK293
- Yeast – Scaling up capabilities up to 10 litres using Wave bioreactor
- Ion Exchange – (cationic and anionic)
- Affinity – (NiNTA, GST, MBP, Protein A, etc.)
- Hydrophobic – (Phenly, Butyl and Octyl sepharose)
- Hydroxyapatite, Size exclusion – (Superdex, Sephacryl, superose)
- Tag removal – On column/off column cleavage Thrombin, Factor Xa, TEV and Enterokinase
- Western blot
- SDS page
- Native page
- Gel Filtration
- Tricine page
- Functional activity assays
GVK BIO has well-established mammalian Cell Culture labs with state-of-the-art infrastructure supporting mini to large scale culture preparations using cell factories. Our vast experience in expressing the drug targets in mammalian systems enables us to express and validate more than 30 GPCRs, various ion channels and transporters.
- We have the capability of performing transient and stable cell line generation.
- We have extensive experience in various human and animal cell lines for stable and transient expression of target proteins.
- Our expertise includes protein expression in HEK293, AD293, CHO, MDCK cell lines.
- We have stably expressed more than 40 functionally active target proteins.
- Our validated stable cell lines include GPCR, ion channels and transporters.
- Expressed proteins are routinely assayed for detection of expression levels and also used for screening potential drug candidates.
Baculovirus-insect Cell Expression Systems
Baculovirus-insect Cell Systems are considered a good system for recombinant glycoprotein production due to the eukaryotic nature of the host. We have established a standard optimisation procedure to determine the conditions for expressing soluble proteins using a Bac-to-Bac expression system.
The variables that we may alter to increase the efficiency and productivity of baculovirus expression are:
- Plaque purification of recombinant viruses.
- Comparison of expression level of different infection parameters: MOI=1, 2, 5 and 10.
- Comparison of expression level of different harvest times: 72, 96, and 120 hours after infection.
- Comparison of expression level of different infection cell densities.
- To summarise, we can provide:
- Functionally active human protein up to 500 mg from E.coli.
- Up to 100 mg of human protein from insect cell culture.
- Stable cell lines expressing protein of interest – 5 billion cells/week.
- Use of cell stackers and cell factories for large number of cell production.
- Use of wave bioreactor for insect Cell Culture.
- Large scale membrane preparation for Radio ligand binding assays.