• Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.

      Bals, Carola; Schambach, Axel; Meyer, Johann; Scheper, Thomas; Rinas, Ursula; Excellence Cluster Rebirth, Institute of Technical Chemistry-Life Science, Leibniz University of Hannover, Callinstr.5, 30167 Hannover, Germany. (2011-03-10)
      Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.
    • Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.

      Li, Zhaopeng; Nimtz, Manfred; Rinas, Ursula; Helmholtz Centre for Infection Research (SB), Braunschweig, Germany. (2011-11)
      Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins ((15)N, (13)C, and (2)H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for (15)N and (13)C, respectively, and 75% of (non-exchangeable) hydrogen for (2)H labeling. The expression yields and final cell densities (OD600 ~16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.
    • Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.

      Tomala, Magda; Lavrentieva, Antonina; Moretti, Pierre; Rinas, Ursula; Kasper, Cornelia; Stahl, Frank; Schambach, Axel; Warlich, Eva; Martin, Ulrich; Cantz, Tobias; et al. (2010-09)
      Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin.
    • Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems.

      Li, Zhaopeng; Kessler, Wolfgang; van den Heuvel, Joop; Rinas, Ursula; Helmholtz Centre for Infection Research (SB), Braunschweig, Germany. (2011-08)
      Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH(4)(+) as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L(-1) product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-D-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.