• 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.
    • Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation.

      Kirschning, Carsten J; Dreher, Stefan; Maass, Björn; Fichte, Sylvia; Schade, Jutta; Köster, Mario; Noack, Andreas; Lindenmaier, Werner; Wagner, Hermann; Böldicke, Thomas; et al. (2010)
      Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (alphaT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. alphaT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence.
    • High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.

      Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2013)
      The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.
    • 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.
    • 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.