Increased Bacterial Cell Density and Recombinant Protein Yield Using a Commercial Microbial Cultivation System

Abstract

EnBase (BioSilta, Finland) is a microbial cultivation system that replicates fed-batch systems through sustained release of glucose by enzymatic degradation of a polymeric substrate. Achievable bacterial cell densities and recombinant capripoxvirus protein expression levels, solubility, and antigenicity using the EnBase system were assessed. BL21-AI Escherichia coli expressing capripoxvirus proteins achieved up to eightfold higher cell densities when grown in EnBase media compared with standard media. Greater yields of capripoxvirus proteins were attained using EnBase media, either through increases in the amount of expressed protein per cell in conjunction with higher cell density or through the increase in cell density alone. Addition of EnBase booster enhanced protein yield for one of the proteins tested but reduced yield for the other. However, the amount of soluble forms of the capripoxvirus proteins tested was not different from that observed from cultures grown under standard conditions. Purified capripoxvirus proteins expressed using EnBase or standard media were assessed for their performance by enzyme-linked immunosorbent assay (ELISA) and were shown to be equally capable of specifically binding capripoxvirus antibodies.

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli

“…These tablets are simply added to sterile water and dissolve in the first minutes of cultivation. Studies have shown that the technology can be easily combined with conventional approaches to yield not only higher amount of a target protein through the higher cell density [ 21 , 25 , 26 ], but also in many cases a higher amount of correctly folded protein per cell [ 26 , 27 ]. Modern screening systems, such as RAMOS, Biolector and PreSens’ SensorDish with an ability for on-line monitoring of growth data provide excellent tools to optimize the growth and expression conditions [ 28 – 32 ].…”

“…As a result, the final product yield is less independent on the time or cell density of induction [ 11 , 21 ]. For example, Peck et al [ 25 ] observed eight times higher cell densities and an increased protein yield per cell producing capripoxyvirus proteins which causes severe diseases in sheep and cattle. The production of Tissue-type plasminogen activators (t-PA) is commonly done in mammalian cell cultures.…”