Escherichia coli as an antibody expression host for the production of diagnostic proteins: significance and expression

Abstract

This review article concerns the production of recombinant antibody fragments for applications mainly in the diagnostic sector. The so-called "point of care diagnostics" is very important for timely diagnosis and treatment, thus being able to save lives and resources. There is intense pressure for more accurate and less expensive rapid diagnostic tests, with a value preferably <$1. Thus, the large-scale cost-effective production of recombinant antibodies is vital. The importance of Escherichia coli toward the production of inexpensive rapid tests will be explained in this review paper. Details about the different strains of E. coli, the strategies used for the insertion and the expression of recombinant proteins, and the challenges that still exist are provided. Afterward, the importance of the expression scale and culture parameters in the final yield of the antibodies are examined. From this analysis, it appears that for good yields of recombinant antibodies, aside from appropriate gene transfer and expression, the culturing parameters are of paramount importance. Larger scale production is more favorable, mainly due to the higher cell densities that can be achieved. Yields of functional Fab fragments in the range of 10-20 mg/L are considered good in shake flasks, whereas in bioreactors can be up to 1-2 g/L. An amount of 10-500 mg of such antibody per million rapid tests is required. Despite the substantial importance of the production of the antibodies and their fragments, their downstream processing should be appropriately considered from the beginning for achieving the target value of the final rapid diagnostic tests.

Engineered multivalent self-assembled binder protein against SARS-CoV-2 RBD

“…In our studies, ACE-MAP yields 74 ± 9 μg/mL (n=3) from 400 mL cultures measured by BCA assay following purification and concentration. E. coli expression systems tend to yield functional Fab fragments in the range of 400-800 μg (in 400 mL culture) [56] using shake flasks indicating that our yield should be improved for a comparable advantage. Thus, expression of our construct needs to further be optimized to provide improved yields whereas E. coli expression systems may often be enhanced with tailored expression time, bioreactor design, temperature, and gene vectors [56] , [57] .…”

“…E. coli expression systems tend to yield functional Fab fragments in the range of 400-800 μg (in 400 mL culture) [56] using shake flasks indicating that our yield should be improved for a comparable advantage. Thus, expression of our construct needs to further be optimized to provide improved yields whereas E. coli expression systems may often be enhanced with tailored expression time, bioreactor design, temperature, and gene vectors [56] , [57] . While ACE-MAP exhibits a strong affinity to SARS-CoV-2 RBD like antibodies, it possesses the advantage of soluble expression in E. coli , and a significantly smaller size – 12 kDa as a monomer (62 kDa as a pentamer).…”

PERISCOPE-Opt: Machine learning-based prediction of optimal fermentation conditions and yields of recombinant periplasmic protein expressed in Escherichia coli

“…The periplasmic expression of recombinant proteins is preferred because the periplasmic space provides an oxidized environment that improves protein folding, especifically for proteins containing di-sulphide bonds. Moreover, the target proteins can be selectively recovered from the periplasmic space using the milder cell disruption steps that avoid the release of cytoplasmic content to the processing fluid [5] , [6] , [7] , [8] . The increasing demands for recombinant proteins have driven the necessity of optimizing various fermentation process parameters to achieve the maximal RPP.…”

Expression and purification of soluble recombinant β-lactamases using Escherichia coli as expression host and pET-28a as cloning vector

“…Many therapeutic proteins and industrial enzymes have been produced for clinical and academic applications using E. coli expression systems, accounting for approximately 30% of the currently approved recombinant proteins [ 7 – 9 ]. It is critical to select a suitable strain for heterologous gene expression based on the characteristics of the available strains and target proteins.…”

“…It is critical to select a suitable strain for heterologous gene expression based on the characteristics of the available strains and target proteins. For example, E. coli K-12 strains and their derivatives produce high levels of acetate, which is detrimental to cell growth and protein expression; thus, these strains are more suitable for propagating recombinant DNA library clones [ 7 ]. E. coli B strains, however, exhibit low acetate accumulation and are commonly used for target protein expression [ 4 ].…”