The ultimate goal of recombinant fermentation research is cost-effective production of desired proteins by maximizing volumetric productivity (to obtain the highest amount of protein in a given volume in the least amount of time). Bioprocessing for recombinant proteins using genetically modified organisms requires a stable, high-yielding recombinant culture, a highly productive fermentation process, and cost-effective recovery and purification procedures. Escherichia coli has been a widely used host for expression of recombinant proteins (1). Its advantages lie in the enormous data available on E. coli cell biology and fermentation process development, as well as its ability to produce large quantities of recombinant proteins cost effectively. Early successful production of insulin by Eli Lilly (www.lilly.com) and bovine growth hormone by Monsanto Corporation (www.monsanto.com) validated the versatility and economic potential of E. coli-based therapeutic protein production. Although it cannot be used to produce complex glycoproteins or proteins with multiple disulfide bonds, over the past 20–30 years E. coli has been used to successfully produce numerous products (2,3).

PRODUCT FOCUS: MICROBIAL PRODUCTS EXPRESSED TO INCLUSION BODY FORM



PROCESS FOCUS: RECOVERY



WHO SHOULD READ: PROCESS DEVELOPMENT, MANUFACTURING



KEYWORDS: TFF, INCLUSION BODIES, MICRO- AND ULTRAFILTRATION, CLARIFICATION, DEPTH FILTRATION, PROTEIN REFOLDING, E. COLI



LEVEL: INTERMEDIATE

Recombinant protein expression using E. coli as a host is frequently associated with the formation of intracellular protein aggregates called inclusion bodies (IBs) (4). The volumetric yield is thus a function of both unit-cell concentration and specific cellular protein yield. Optimization of high-cell-density fed-batch fermentation process is a key step in enhancing the volumetric yield of recombinant proteins (5). High-level expression of a protein of interest in IBs facilitates its physical isolation from the cytoplasm at the cost of its native structure. Renaturation to its bioactive form is cumbersome and provides low recovery of the final product. It also accounts for the major cost in overall production of recombinant proteins from microbial systems (6). However, for cases in which a simple, high-yielding protein refolding process is developed for an aggregated recombinant protein, high-level expression of it as IBs provides a straightforward strategy for cost-effective production and purification. Thus, in spite of problems associated with the IBs in E. coli, the system has been extensively used in commercial therapeutic protein production.

Recombinant Proteins As IBs

Inclusion bodies are dense particles of aggregated protein found in both the cytoplasmic and periplasmic space when E. coli expresses foreign proteins at high levels. IB size varies 0.5–1.3 µM, and IB protein aggregation may be either amorphous or paracrystalline depending on localization (7). In many cases, IBs constitute 20–50% of the total cellular protein in a bacterium.

In addition to heterologous proteins, IBs contain very small amounts of host protein, ribosomal components, and DNA/RNA fragments. It has also been reported that the presence of contaminants in isolated IBs is mainly due to incomplete purification of them following cell lysis (8), but it could result from accidental trapping during the aggregation of polypeptide into IB form. IBs often contain the overexpressed protein almost exclusively, and such aggregation has been reported to be reversible (8).

Formation of inclusion bodies thus facilitates the easy isolation and recovery of expressed proteins in a denatured form. Because IBs have high density (∼1.3 mg/mL), they are easily separated by high-speed centrifugation after cell disruption. The most efficient process for complete cell lysis is high-pressure disruption. Lysozyme is used rarely in large-scale processes because of its cost and regulatory concerns about animal-sourced material. Homogenization is more often performed using mild detergents, although lysozyme is still used in some processes developed some time ago.

Further purification can be achieved by washing with detergents, a low concentration of salt, and/or urea. With proper isolation and washing, IBs of >95% purity can be prepared from E. coli. Some studies indicate that sucrose-gradient centrifugation can provide very pure IB preparations; however, preparative gradients are not scalable or cost effective for production. Ultrafiltration with membranes of different pore size has also been used for IB isolation from E. coli cells (9).