Production of protein in E. coli (Escherichia Coli) and purification

Proteins production protocol:

Step 0: Gene synthesis and codons optimization

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This gene synthesis step is very important because it allows us to optimize rare codons for E. coli expression system. It significantly increases production yields in many cases and especially with E. coli. This strategy has proven to be even more relevant when several runs of production are needed or when large amounts of protein are needed. A quick look at the cDNA sequence allows to identify if the sequence contains a high content of rare codons that the bacteria will have a hard time to translate from mRNA into protein.

Step I: cDNA cloning into an protein expression vector

This step consists in integrating the cDNA into a bacterial expression vector containing a purification tag and inducing the expression of the fusion protein of interest and allowing the protein purification on affinity column via the tag. The tag can be removed if necessary for functional studies.

Step II: Production of protein evaluation:

Escherichia coli colonies

  • Transformation in different bacterial strains
  • Bacterial cultures in different conditions (medium types, additives, IPTG concentration, temperature, etc.)
  • Synthesis induction (kinetic of 5 hours)
  • Affinity purification with a specific resin
  • Acrylamide gel (SDS-PAGE) and/or Western blot analysis

NB: We perform several runs of tests in order to identify optimal conditions:

  • of culture for the protein production
  • of protein extraction (soluble or in inclusion bodies) necessary for the recombinant protein folding
  • of protein purification
  • of production yields

In order to identify the optimal conditions for the synthesis of proteins, we need to modify a large number of parameters and we are involved in all stages including codons optimization, vector choice, tag, culture medium, additives, bacterial strains, induction temperatures, IPTG concentrations, purification technique types, renaturation / refolding (dialysis buffer change)… As a leading manufacturer protein, we countinuously strive to improve our processes and it is one of the main strengths of our organization.

Step III: Production of proteins and purification scale up:

  • Bacterial culture: 1 or more liters
  • Growing conditions according to the optimized protocol established during step II
  • Preparation of bacterial lysates according to the optimized protocol established during step II
  • Affinity chromatography purification according to the optimized protocol established during step II
  • Optional: tag removal by enzymatic cleavage

NB: the production step is carried out according to customer specifications based on results obtained at the end of step II. Many kinds of purification types are available including tag affinity purification (His, GST, MBP, Sumo), size exclusion chromatography, ion exchange, etc.

Turnaround time :

  • Cloning: 2 weeks
  • Expression evaluation: 10 days
  • Production and purification: 5 days

ProteoGenix – production of recombinant proteins in E. Coli

ProteoGenix has extensive experience in the custom production of proteins in Escherichia coli. We are able to produce any type of protein. Our experience in this industry gives us a large number of solutions and technical advantages that allows us almost always to obtain the required material to:

  • develop your polyclonal or monoclonal antibodies
  • conduct immunogenicity studies
  • perform structural or crystallography studies
  • conduct functional analysis
  • build your assays tools (standard, positive control)
  • perform Western blot experiments
  • perform immunoprecipitation experiments
  • conduct ELISA experiments
  • conduct dot-blot experiments, etc.

We also accept protein synthesis orders for very large scale production in fermentors.

Large Scale expression of protein in E. Coli

E. coli protein production enables to reach high expression yields, at low cost and within a very short period of time because the growth rate of bacteria is much faster than any other system. However, this system is not appropriate when post-translational modifications (PTM) are necessary for the required application.