Recombinant protein expression in Escherichia coli: advances and challenges (1)
In glucose-salts media and given the optimal environmental conditions, E.coli’s doubling time is about 20 min. The theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight/l or roughly 1 × 1013 viable bacteria/ml. Transformation with exogenous DNA is fast and easy. Plasmid transformation of E. coli can be performed in as little as 5 min.
The most common expression plasmids in use today are the result of multiple combinations of replicons, promoters, selec- tion markers, multiple cloning sites, and fusion protein/fusion protein removal strategies. The use of high copy number plasmids for protein expression by no means implies an increase in production yields. The T7 promoter system present in the pET vectors (pMB1 ori, medium copy number) is extremely popular for recombinant protein expression. This is not surprising as the target protein can represent 50% of the total cell protein in successful cases.
A mutant λcI repressor protein ( λcI857) is temperature-sensitive and is unstable at temperatures higher than 37◦C. E. coli host strains containing the λcI857 pro- tein (either integrated in the chromosome or into a vector) are first grown at 28–30◦C to the desired density, and then protein expression is induced by a temperature shift to 40–42◦C.
In the E. coli system, antibiotic resistance genes are habitually used for this purpose. Resistance to ampicillin is conferred by the bla gene whose product is a periplasmic enzyme that inactivates the β-lactam ring of β-lactam antibiotics.
It is invaluable to have means to (i) detect it along the expression and purification scheme, (ii) attain maximal solubility, and (iii) easily purify it from the E. coli cellular milieu. The expression of a stretch of amino acids (peptide tag) or a large polypeptide (fusion partner) in tandem with the desired protein to form a chimeric protein may allow these three goals to be straightforwardly reached. Common examples of small peptide tags are the poly-Arg-, FLAG-, poly-His-, c-Myc-, S-, and Strep II- tags. On the other hand, adding a non-peptide fusion partner has the extra advantage of working as solubility enhancers. Enterokinase and thrombin were popular in the past but the use of His-tagged TEV has become an everyday choice due to its high specificity, it is easy to produce in large quantities and leaves only a serine or glycine residue (or even the natural N-terminus) after digestion. The BL21(DE3) and its derivatives are by far the most used strains for protein expression. High throughput protocols adapting automatic liquid handling robots have been described, making it possible for a single person to test more than 1000 culture conditions within a week.
If the growth rate of the recombinant strain is slower compared to an empty-vector bearing strain then two causes may explain the phenotype: gene toxicity and basal expression of the toxic mRNA/protein. After control of basal expression, the culture should grow well until the proper time of induction. At this moment, if the protein is toxic, cell growth will be arrested. In many cases, the level of toxicity of a protein becomes apparent when a certain threshold of host tolerance is reached and exceeded. To remove the protein from the cell, the signal peptides of the following proteins are widely used for secretion: Lpp, LamB, LTB, MalE, OmpA, OmpC, OmpF, OmpT, PelB, PhoA, PhoE, or SpA.
If codon bias could be an issue when expressing a recombi- nant protein, a large number of free online apps detect the presence of rare codons in a given gene when E. coli is used as a host (molbiol.ru/eng/scripts/01_11.html). Some are freely available as web servers or standalone software.
Despite being a rich broth, cell growth stops at a relatively low density. Increasing the amount of peptone or yeast extract leads to higher cell densities. Also, divalent cation supplementation (MgSO4 in the millimolar range) results in higher cell growth. More agitation is generated in baffled flasks; under these con- ditions, 350–400 rpm are enough for good aeration. However, vigorous shaking can induce the formation of foam, which will lower oxygen transfer. the culture volume should be less or equal to 10% of the shaking flask capacity, although in our hands, protein production with culture volumes occupying 20% of the flask capacity was possible.
IB formation results from an unbalanced equilibrium between protein aggregation and solubilization. So, it is possible to obtain a soluble recombinant protein by strategies that ameliorate the factors leading to IB formation. The formation of erroneous disulfide bonds can lead to protein misfolding and aggregation into IB. Disulfide bond formation at these sites may lead to protein inactivation, misfolding, and aggregation. When proteins are purified from IB, urea-denatured and then refolded in vitro, addition of osmolytes (also called chemical chaperones) in the 0.1–1 M range of concentration increases the yield of soluble protein. When IB formation is a problem, recombinant protein synthe- sis should be carried out in the range 15–25◦C. When working at the lower end of the temperature range, slower growth and reduced synthesis rates can result in lower protein yields.
