Protein Expression Protocol & Troubleshooting in E. coli

Protein expression in E. coli is a powerful and cost-effective way to produce large quantities of purified protein for biochemical, structural, and therapeutic applications. Boster Bio offers a full range of recombinant protein expression services in both E. coli and mammalian cell cultures with competitive price and fast turnaround time to meet your needs on the production of recombinant proteins from a variety of organisms. 

Boster Bio has extensive experience in high-throughput gene synthesis, cloning, mutagenesis, protein purification, and antibody production. BosterBio provides one-stop recombinant protein expression service, and provides clients with a complete solution for their protein expression needs including high quality protein expression services, timely delivery and reasonable price.

E Coli Protein 

  1. coli is a gram-negative bacterium that is often used as a host for protein expression. It is a model organism, which means that it can be manipulated easily and studied in detail. E. coli grows well at room temperature, so it must be grown in an incubator at 37˚C with shaking every 10 minutes to keep the cells aerated. Because E. coli is easy to handle and manipulate, it has been used as the model organism for many important discoveries about genetics and protein production by recombinant DNA technology. Efficient protein expression service systems have been developed to allow large quantities of proteins to be produced quickly, cheaply and reproducibly. These systems have been adapted for use with various hosts including mammalian cells, yeast and plants.
  2. coli Protein Expression Service at Bostser Bio

If you are looking to generate your own recombinant protein, Bostser Bio is the place to go. We provide one-stop recombinant protein expression service and have a group of experienced scientists who are proficient in various methods such as baculovirus expression system, mammalian cell culture system and E. coli expression system. Boster Bio products are of high quality and are reliable. Boster Bio protein expression services are one of the best in the industry. 

  1. coli Protein Expression Advantages
  2. coli is a Gram-negative bacterium, and as such has a cell wall with an outer membrane. The outer membrane contains porins that allow the passage of small molecules into the periplasmic space. E coli has several advantages including:
  • E. coli can grow on minimal media containing sugars and amino acids but lacking vitamins or other organic compounds. 
  • E coli is often used in molecular genetics as it is easy to culture, fast growing under optimal conditions, inexpensive compared to other lab organisms well-understood scientifically, and can be stored at -80 °C almost indefinitely.
  • E. coli has a short turnaround time (only 2 weeks),
  • E coli has a stable supply (1 mg is enough for most biochemical studies), 
  • E coli has a high transformation efficiency (10^8 CFU/μg pUC19) which is especially important for high throughput projects such as compound screening or antibody engineering

Recombinant protein expression in E. coli

Recombinant protein expression in E. coli is a common practice in biochemistry and molecular biology. The bacterium Escherichia coli is a gram-negative prokaryotic organism that grows under many different conditions, thus making it very amenable to genetic manipulation. In addition, its fast growth rate makes it well suited for large scale protein production in bacterial cultures that can be used as cell factories or fed into downstream processes such as purification and formulation.

  1. coli is also known as the “friendly” bacteria because it does not cause disease in humans (or animals) like other bacteria do; some strains of E. coli even live inside our intestines. There are several different species of E. coli.

Troubleshooting for recombinant protein expression in E. coli

It is common to have a problem in the protein expression process. The following are some troubleshooting steps you can take to fix the problem:

  • Check your media and buffer conditions, to ensure that the nitrogen source in your media is adequate for your strain of E. coli and that it matches those you’ve used previously for this type of experiment. 
  • If you are using a different nitrogen source, make sure the pH is within range (5-8) and check its concentration on an analytical balance with minimal error (<0.01). 
  • Make sure there are enough carbon sources (usually glucose) in your growth medium so that your bacteria do not starve when they can’t express recombinant proteins as fast as they should be growing (or at all). 
  • If necessary, add more carbon sources or reduce their concentrations slightly if they appear too high relative to other nutrients like nitrogen or phosphate which will likely cause problems with growth if present at too large amounts relative to carbon sources such as glucose. 

Summary of protein eukaryotic gene expression in E. coli

We have looked at how to troubleshoot E coli and what  E coli protein is, and we can summarise the steps protein eukaryotic gene expression in E. coli thus:

Convert your DNA sequence into a cDNA library using PCR and dNTPs

Transform your cDNA library into a plasmid vector by electroporating it into electrocompetent cells; transformants are selected for on ampicillin-containing plates and then plated onto LB agar containing ampicillin to ensure that only cells that have taken up the new plasmid are grown

Grow your transformed overnight at 37°C with shaking so that you get large colonies on your plate (if no colonies appear, check if there were any transformants)

Conclusion

In Conclusion, recombinant protein expression in E. coli is the most extensively used approach to produce functional proteins in vitro. Boster Bio makes use of a high-throughput system to provide high quality services and cost-effective production of customer’s protein of interest. Boster also provide assistance with troubleshooting if your project is not going as planned, such as clone screening or modifying the cloning strategy that was used.

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