High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization

The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria–Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.Fil: Kangwa, Martin. Jacobs University; AlemaniaFil: Yelemane, Vikas. Jacobs University; AlemaniaFil: Polat, Ayse Nur. Jacobs University; AlemaniaFil: Gorrepati, Kanaka Durga Devi. Jacobs University; AlemaniaFil: Grasselli, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Fernández Lahore, Marcelo. Jacobs University; Alemani

Improved biomass and protein production in solid-state cultures of an Aspergillus sojae strain harboring the Vitreoscilla hemoglobin

The biotechnological value of Aspergillus sojae ATCC 20235 (A. sojae) for production of pectinases in solid-state fermentation (SSF) has been demonstrated recently. However, a common drawback of fungal solid-state cultures is the poor diffusion of oxygen into the fungi that limits its growth and biological productivity. The bacterial Vitreoscilla hemoglobin (VHb) has favored the metabolism and productivities of various bacterial and yeast strains besides alleviating hypoxic conditions of its native host, but the use of VHb in filamentous fungi still remains poor explored. Based on the known effects of VHb, this study assessed its applicability to improve A. sojae performance in SSF. The VHb gene (vgb) under control of the constitutive Aspergillus nidulants gpdA promoter was introduced into the genome of A. sojae by Agrobacterium-mediated transformation. Successful fungal transformants were identified by fluorescence microscopy and polymerase chain reaction (PCR) analyses. In solid-state cultures, the content of protease, exo-polygalacturonase (exo-PG), and exo-polymethylgalacturonase (exo-PMG) of the transformed fungus (A. sojae vgb+) improved were 26, 60, and 44 % higher, respectively, in comparison to its parental strain (A. sojae wt). Similarly, biomass content was also 1.3 times higher in the transformant strain. No significant difference was observed in endo-polygalacturonase (endo-PG) content between both fungal strains, suggesting dissimilar effects of VHb towards different enzymatic productions. Overall, our results show that biomass, protease, and exo-pectinase content of A. sojae in SSF can be improved by transformation with VHb.Fil: Mora Lugo, Rodrigo. Jacobs University; AlemaniaFil: Madrigal, Marvin. Jacobs University; Alemania. Universidad de Costa Rica; Costa RicaFil: Yelemane, Vikas. Jacobs University; AlemaniaFil: Fernandez Lahore, Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Jacobs University; Alemani

Growth-dependent surface characteristics of Hansenula Polymorpha: implications for expanded bed adsorption chromatography

The cell surface characteristics of a methylotrophic wild-type strain of yeast, Hansenula polymorpha, was investigated at different growth stages (early log, late log, stationary and death) of the biomass under different conditions (low and high salt in intact and disrupted forms) using extended DLVO theory. Biomass was characterized by contact angle measurements as well as zeta potential determinations. These measurements were used to describe the hydrophobic, polar, and electrostatic behavior of the biomass in its growth stages. Consequently, interaction free energy vs. distance profiles of the biomass with anion-exchange and HIC adsorbents were conveniently generated. A strong interaction was calculated between cells and the adsorbents in the stationary and death phases of the biomass illustrated by the striking correlation between theoretical predictions and biomass deposition experiments. The physico-chemical properties of biomass in different growth phases have important implications for expanded bed adsorption chromatography, where unfavorable biomass-adsorbent interactions adversely affect process efficiency.Fil: Naz, Nadia. Jacobs University; AlemaniaFil: Dsouza, Roy N.. Jacobs University; AlemaniaFil: Yelemane, Vikas. Jacobs University; AlemaniaFil: Vennapusa, Rami Reddy. Shantha Biotechnics; IndiaFil: Kangwa, Martin. Jacobs University; AlemaniaFil: Fernandez Lahore, Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Jacobs University; Alemani

MOESM1 of High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization

Additional file 1: Figure S1. DNA nucleotide sequence for Avipure (underlined). Start codons is indicated in bold. Stop codon is highlighted in red