Further, intracellular overexpression of recombinant proteins in bacteria often leads to the formation of insoluble aggregates, or inclusion bodies. coli required mutation of an internal cysteine to ensure proper disulfide bond formation after cell lysis. Though this process is used for many products at industrial scale-including insulin, interleukin-2 (IL-2), and human growth hormone (hGH)-intracellular expression introduces product and process development challenges. A typical production process involves intracellular product expression, which requires cell lysis and multi-step purification to isolate the product from the cellular contents. coli, are favored as bacterial hosts for protein production-they can express a broad range of heterologous proteins at high titers and are robust in industrial-scale culture. Progress in engineering these and other Gram-positive hosts for protein production is well-summarized in several review articles. Nevertheless, the range of compatible proteins remains narrow, and growth of filamentous organisms such as S. lividans has low extracellular protease activity while retaining a high secretion capacity. Compatibility with heterologous proteins is largely limited to enzymes, however, and extracellular proteolytic degradation is a significant product quality issue. subtilis has a high secretion capacity-titers on the order of grams per liter have been achieved. Secretion stress response systems and folding chaperones provide extracellular quality control. Gram-positive bacteria such as Bacillus subtilis and Streptomyces lividans allow protein secretion directly to the extracellular medium through the general secretory pathway, as they lack an outer membrane (OM). īacterial hosts offer fast growth, relative genetic simplicity, and genetic stability. Yeasts typically are faster growing but can suffer from genetic instability and clonal variation. Mammalian cells, however, are slow growing, expensive to culture, have large batch to batch variations, and can be difficult to genetically engineer. Eukaryotic hosts such as Saccharomyces cerevisiae, Pichia pastoris, Chinese hamster ovary (CHO) cells, or HEK 293 cells secrete recombinant proteins through their native secretion machinery at high titers. Secreting protein to the extracellular space increases initial purity and thus can decrease the complexity of downstream processing. These vast differences in physicochemical properties call for a variety of strategies to ensure that production of heterologous proteins is robust and scalable. In recent years, the chemical and structural diversity of protein products has expanded rapidly, with targets such as short anti-microbial peptides, antibody-like binding proteins for diagnostics, and protein biomaterials. The development of recombinant insulin and its production in Escherichia coli in the 1980s launched an industry that comprises billion dollar markets, with products ranging from protein biologics ($91B) to industrial enzymes ($4.8B). This review will cover recent advances toward the development of high-expressing, high-secreting Gram-negative production strains. An additional focus is the expression of secretion machineries from pathogenic bacteria in the “workhorse” of biotechnology, Escherichia coli, to reduce handling of pathogenic strains. Efforts include modifying the secretion tags required for recombinant protein secretion, developing methods to screen or select rapidly for clones with higher titer or efficiency, and improving reliability and robustness of high titer secretion through genetic manipulations. Building on past successes, researchers continue to increase the secretion efficiency and titer through these systems in an effort to make them viable for industrial production. Secretion from these bacteria operates through highly specialized systems, which are able to translocate proteins from the cytosol to the extracellular medium in either one or two steps. Thus, researchers are devoting significant attention to engineering Gram-negative bacteria to secrete recombinant proteins to the extracellular medium. Secretion of biotechnologically relevant proteins into the extracellular medium increases product purity from cell culture, decreases downstream processing requirements, and reduces overall cost. However, the utility of these microbes would expand if they also could secrete the product at commercial scales. Gram-negative bacteria are attractive hosts for recombinant protein production because they are fast growing, easy to manipulate, and genetically stable in large cultures.

Gram negative bacteria membrane