Obtaining a pure, highly concentrated (mM) protein sample is a major bottleneck for both x-ray crystallography and NMR. The high concentration is required because both techniques are insensitive to single molecule analysis, and a large population of a particular protein is required to overcome the signal-to-noise barrier. On a similar note the sample needs to be very homogenous, so protein purification is necessary at some point.
Recombinant protein production using E. coli is the method of choice when large quantities of protein are required. This process involves taking the gene (often cDNA) of the protein of interest, splicing it into a suitable inducible vector, transforming the vector into an E. coli host, and growing the culture in a rich medium. The bacterial host will multiply during a growth phase, after which it is induced to express the protein of interest. If all goes well, the protein will express solubly and in high numbers. Unfortunately, this process is easier said than done. Many eukaryotic proteins do not express well in prokaryotic hosts, and oftentimes modifications need to be made to optimize the bacterial host, codon usage, media, etc. to obtain a decent yield of recombinant protein. Additionally, proteins often express insolubly as inclusion bodies and require high concentrations (2M to 8M) of denaturants such as urea or guanidine hydrochloride to solubilize them, and then stepwise dialysis into an appropriate buffer to refold them. Alternatively, eukaryotic organisms such as S. cerevisiae (yeast), insect and mammalian cell lines can be used, especially when post-translation modifications are required, though a decrease in yield and increase in overall cost is common with these organisms.
Protein Production in E. coli
The gram negative bacteria, E. coli is the first choice expression host for the production of recombinant proteins and protein complexes from prokaryotes and can be used to produce eukaryotic proteins particularly the structural/functional domains of larger proteins. The advantages of using E. coli include the ease of handling, short cell doubling time leading to rapid production of samples and the low cost of scaling up to multi-litre volumes to produce high yields of proteins.
Protein Production in insect cells
The baculovirus expression vector system (BEVS) is a powerful eukaryotic method to produce proteins and protein complexes. BEVS uses a recombinant baculovirus carrying the heterologous genes of interest, to infect insect cell cultures provided as monolayers or suspension. BEVS has become particularly attractive for protein research as it combines the ability to produce difficult-to-express, eukaryotic proteins with high yields and often authentic processing (PTMs) with simple cultivation needs without particular safety precautions. Important technological advances have greatly improved upon the original procedures for the generation of recombinant baculoviruses which were time consuming and required specialised expertise.