Production of Active Mammalian and Viral Proteases in Bacterial Expression Systems

Yeast-Based Screening of Anti-Viral Molecules

“…For example, the expression of proteases from HIV [ 126 ], poliovirus [ 127 ], hepatitis A [ 128 ], and SARS-CoV [ 103 ] has been found lethal for cells. Like eukaryotic cells, the expression of viral protease is toxic even to bacterial cells [ 129 ], and this further complicates the development and application of in vivo assays for the screening of viral protease inhibitors. Because of these complications, in vitro assays are more common for screening anti-viral molecules.…”

Direct production of functional matrix metalloproteinase—14 without refolding or activation and its application for in vitro inhibition assays

“…In addition to providing an oxidative environment that facilitates the formation of disulfide bonds, periplasmic expression can enhance proper protein folding due to multiple molecular chaperons (e.g., SurA, PpiA, PpiD, FkpA, and Skp) (Baneyx and Mujacic, ) and a slow processing rate controlled by secretion machineries (Wülfing and Plückthun, ). Following successful overexpression of T cell receptor fragments in E. coli periplasm (Ward, ), many recombinant proteins such as antibody fragments and bacterial, viral and mammalian proteases have been periplasmically produced (Babé et al, ; Frenzel et al, ; Harvey et al, ; Makino et al, ; Sroga and Dordick, ). In most of these studies, the mammalian proteases were expressed as pro‐enzymes or fused with DsbC, GST, or OmpT leader sequence, and therefore additional steps were needed for activation or fusion partner removal (Babé et al, ).…”

“…Following successful overexpression of T cell receptor fragments in E. coli periplasm (Ward, ), many recombinant proteins such as antibody fragments and bacterial, viral and mammalian proteases have been periplasmically produced (Babé et al, ; Frenzel et al, ; Harvey et al, ; Makino et al, ; Sroga and Dordick, ). In most of these studies, the mammalian proteases were expressed as pro‐enzymes or fused with DsbC, GST, or OmpT leader sequence, and therefore additional steps were needed for activation or fusion partner removal (Babé et al, ). Previously, human MMP‐14 with its propeptidic domain (Pro‐MMP‐14) has been periplasmically expressed, followed by chromatographic purification and treatments with 4‐aminophenylmercuric acetate (APMA) or trypsin to yield the functional enzyme (Will et al, ).…”

Proteasas digestivas de tipo tripsina del taladro del maíz, "Sesamia nonagrioides" (Lepidoptera, Noctuidae), caracterización e interacción con la proteína insecticida Cry1Ab

“…Existen numerosos trabajos realizados sobre producción de proteasas en diversos sistemas de expresión, lo que indica que las proteasas pueden ser expresadas en la mayoría de los sistemas de expresión utilizados actualmente (Babé et al, 2000;Gasparian et al 2003;Hohenblum et al, 2004). Así, se han expresado tripsinas adaptadas al frío específicas del pez Tautogolabrus adspersus, en P. pastoris (Macouzet et al, 2005), una proteasa termoestable con temperaturas óptimas entre 85-90º C (termopsina) específica de arqueobacterias termófilas en E. coli y en células de insectos mediante baculovirus (Lin et al, 1992), carboxipeptidasa B de porcino en P. pastoris (Ventura et al, 1999), tripsinas de rata en E. coli (Vasquez et al, 1989;Yee y Blanch, 1992), tripsina de bovino en plantas de maíz (Woodard et al, 2003) y catepsina C del nemátodo Schistosoma japonicum en células del lepidóptero Trichoplusia ni mediante el uso de baculovirus (Hola-Jamriska et al, 2000).…”

“…A pesar de las dificultades que presenta el sistema de expresión de E. coli, para producir proteasas funcionales de manera heteróloga, se han expresado con éxito este tipo de proteínas en numerosas ocasiones (Babé et al, 2000). Se han conseguido renaturalizar mediante varios métodos como dilución, diálisis o cromatografía (Sorensen y Mortensen, 2005).…”