Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production

Abstract

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.

Study of kinetic parameters for the production of recombinant rabies virus glycoprotein

“…Recently, we identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of rabies virus glycoprotein, expressed in Drosophila melanogaster cells, by about 60% (Mendonça et al 2008). This work aimed to extend the previous work by determining the kinetic parameters for cell growth and recombinant protein production (i.e, maximum specific rates-l Xmax and l rRVGPmax ) and the relationship between the production of the recombinant protein and the phase of the cell cycle on the addition of the hemolymph.…”

“…These procedures aim to allow high cellular densities at low cost and with reduced operation complexity. Several studies have reported the presence of substances with pharmacological activity in the insect hemolymph (Shiotsuki et al 2000;Yamamoto et al 1999;Guerrero et al 1999;Jiang et al 1999;Rosenfeld and Vanderberg 1998;Hamdaoui et al 1998;Maranga et al 2003;Huberman et al 1979;Lin et al 1998;Kurata et al 1994;Jones et al 1993;Zhu et al 2000;Lowenberger et al 1999;Lamberty et al 1999;Gross et al 1998;Johns et al 1998;Lanz-Mendoza et al 1996;Peters et al 1993;Souza et al 2005;Raffoul et al 2005; Kanaya and Kobayashi 2000;Mendonça et al 2008). Kanaya and Kobayashi (2000) isolated and characterized a protein from silkworm hemolymph able to increase the activity of a recombinant protein (luciferase) by approximately 6,000 times.…”

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of Lonomia obliqua

“…We have identified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59% (Mendonça et al 2008). The highest values of RVGP production were observed in the exponential phase The protein concentration was semi-quantified by densitometry expression system was used to verify the beneficial effects of hemolymph in this recombinant protein production system.…”

“…However, baculovirus infection induces oxidative stress and cell death, which limits recombinant protein production (Vieira et al 2006). Recently some studies have demonstrated the presence of pharmacologically active substances in hemolymph of Lonomia obliqua, which can enhance Sf-9 insect cell growth and culture longevity (Maranga et al 2003;Raffoul et al 2005;Souza et al 2005;Mendonça et al 2008;Greco et al 2009). In the present work, hemolymph (whole extract or a semi-purified fraction) was applied to improve BEVS/IC system by increasing rotavirus recombinant protein.…”

Kinetic studies of recombinant rabies virus glycoprotein (RVGP) cDNA transcription and mRNA translation in Drosophila melanogaster S2 cell populations

“…Several complex glycoproteins were already expressed in the S2 cell system, using these promoters (Mallender et al 2001;Zhang et al 2007;Scotter et al 2006;Brillet et al 2006;Kim et al 2005;Johansson et al 2007;Jennings et al 2006;Li et al 2005;Lim et al 2004;Lee et al 2007). Aiming the expression of high levels of RVGP under the control of these promoters in the S2 cell system, for vaccination as well as structure/function evaluation, many studies were already carried out on cell growth and heterologous recombinant protein expression kinetics (Yokomizo et al 2007;Galesi et al 2008;Swiech et al 2008a;Batista et al 2009;Ventini et al 2010;Lemos et al 2009), as well as on metabolism and synthesis of secondary products (Swiech et al 2008b, c) and culture medium formulation and supplementation (Galesi et al 2007;Batista et al 2008Batista et al , 2011Mendonça et al 2008Mendonça et al , 2009. For constitutive RVGP expression using the actin promoter, instead of a gradual and sustained increase of RVGP, we have observed a sharp RVGP increase, at the beginning of the stationary cell growth phase, which could not be associated with the culture system or the cell culture media, pH, oxygen concentration or substrate change (Galesi et al 2008;Ventini et al 2010;Batista et al 2011).…”