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Making soluble recombinant proteins from Escherichia coli: Lessons learned from the human interferon alpha
Abstracts / Journal of Biotechnology 131S (2007) S69–S72
5. Making soluble recombinant proteins from Escherichia coli: Lessons learned from the human interferon alpha Imen Rabhi-Essafi ∗ , Noureddine Khalaf, Amine Sadok, Dahmani M. Fathallah Institute Pasteur of Tunis, BP74, Le belvedere, Place Pasteur, 102 Tunis, Tunisia Most of the biopharmaceuticals or bio-drugs available in the market are recombinant proteins and most of the marketed pharmaceutical grade recombinant proteins are produced and purified from E. coli. However, protein expressed in large amount in E. coli often precipitate into insoluble aggregates called inclusion bodies that are, in general, misfolded proteins and thus biologically inactive. Refolding from inclusion bodies is undesirable, because of the poor recovery yield and the requirement for optimization of the refolding conditions for each target protein. The purification of soluble recombinant proteins is more cost effective and less time consuming. Hence solubility is a key issue for the production of recombinant protein in heterologous expression systems. Soluble recombinant proteins are often properly folded, functional, and easier to purify than aggregated proteins from inclusion bodies. To achieve highlevel expression of soluble and functional recombinant human IFN␣ (hIFN␣) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFN␣2b) in which we merged the human IFN␣2b cDNA with the Glutathione-S-transferase coding sequence downstream of the inducible tac promoter. Using this plasmid, we have achieved 70% expression of soluble recombinant hIFN␣2b (rhINF␣2b) as a GST fusion protein using the E. coli BL21 strain, after optimization of environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codons within the thrombin cleavage site. We have used the engineered plasmid (pGEX--hIFN␣2b) and the modified E. coli trxB− /gor− (Origami) strain to overcome the problem of removing the GST moiety and to over-express and purify the rhIFN␣2b. Our results show the production of soluble and functional rhIFN␣2b at a yield of 100 mg/L, without optimization of any step of the process. The antiviral specific activity of the purified soluble IFN␣2b varied from 1.1 × 108 to ∼2.0 × 108 IU/mg as compared to the WHO IFN␣ standard. Our data show that production at high yield of a soluble and functional rhIFN␣2b can be achieved in E. coli when the protein is tagged with GST, and upon engineering of the GST-IFN junction this strategy can be applied to other proteins of medical interest. doi:10.1016/j.jbiotec.2007.07.123
S71
6. Cell stage-specific alpha-dystrobrevin distribution in subcellular compartments and association with other proteins in human cancer cells Veronika Borutinskaite a,∗ , Ruta Navakauskiene b , Karl-Eric Magnusson a a
Linkoping University, Veronika Borutinskaite, medicinsk mikrobiologi, Halsouniversitetet, Linkoping SE 581 85, Sweden b Institute of Biochemistry, Vilnius, Lithuania The dystrophin-associated protein complex (DPC) is expressed in muscle and non-muscle tissues. It is considered to be not only a mechanical component of cells, that links the cortical actin cytoskeleton to the extracellular matrix, but also a highly dynamic multifunctional structure that can serve as a scaffold for signaling proteins. Part of the signaling function of the DPC may be mediated by the dystrobrevins (DB). The involvement of DB in signaling pathways depends on the interaction with other proteins, like dystrophin, syntrophin, sarcoglycan complex and actin. In this study we have studied the possible role of alpha-DB in human non-muscle cancer cells (NB4 and HeLa). Analysing DB isoform expression in different cell fractions, total hydrophilic and hydrophobic proteins were isolated from proliferating NB4 cells as well as from cell nuclei only. We found that the alpha-DB isoforms were expressed differently in the NB4 cell fractions. We demonstrated that the hydrophilic fraction contained all major alpha-DB isoforms. In contrast, only alpha-DB-1 (77 kDa) was detected in the hydrophobic fraction of either cellular or nuclear membranes; this isoform could be important for cytoskeleton reorganisation during cell proliferation. Retinoids have been extensively studied over the years, and it has seen that all trans-retinoic acids (ATRA) likely provide the best example of the successful use in the treatment of acute promyelocytic leukemia (APL). Recently we have shown that combinations of ATRA with specific histone deacetylase inhibitors (HDACI) have synergistic effects on leukemia cell differentiation in vitro. Moreover, HDACI affect cancer cell division, induce differentiation and/or apoptosis (Savickiene et al., 2006). It has also been shown that alpha-DB undergoes phosphorylation which can be important in signal transduction (Grady et al., 2003). Using immunoprecipitation, we determined alphaDB phosphorylation after treatments with HDACI, BML-210 and ATRA. We found changes in tyrosine phosphorylation of alpha-DB-2 after treating NB4 cells with ATRA and BML210. In contrast, a combination of ATRA and BML-210 did not effected the phosphorylation level neither in the cytoplasmic nor in the nuclear fraction. With mass spectrometry analysis, new proteins interacting with alpha-DB were discovered. Confocal microscopy was used for visualization of alpha-DB localization in the cell, where colocalizations with actin and Hsp90 were established.
5. Making soluble recombinant proteins from Escherichia coli: Lessons learned from the human interferon alpha Imen Rabhi-Essafi ∗ , Noureddine Khalaf, Amine Sadok, Dahmani M. Fathallah Institute Pasteur of Tunis, BP74, Le belvedere, Place Pasteur, 102 Tunis, Tunisia Most of the biopharmaceuticals or bio-drugs available in the market are recombinant proteins and most of the marketed pharmaceutical grade recombinant proteins are produced and purified from E. coli. However, protein expressed in large amount in E. coli often precipitate into insoluble aggregates called inclusion bodies that are, in general, misfolded proteins and thus biologically inactive. Refolding from inclusion bodies is undesirable, because of the poor recovery yield and the requirement for optimization of the refolding conditions for each target protein. The purification of soluble recombinant proteins is more cost effective and less time consuming. Hence solubility is a key issue for the production of recombinant protein in heterologous expression systems. Soluble recombinant proteins are often properly folded, functional, and easier to purify than aggregated proteins from inclusion bodies. To achieve highlevel expression of soluble and functional recombinant human IFN␣ (hIFN␣) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFN␣2b) in which we merged the human IFN␣2b cDNA with the Glutathione-S-transferase coding sequence downstream of the inducible tac promoter. Using this plasmid, we have achieved 70% expression of soluble recombinant hIFN␣2b (rhINF␣2b) as a GST fusion protein using the E. coli BL21 strain, after optimization of environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codons within the thrombin cleavage site. We have used the engineered plasmid (pGEX--hIFN␣2b) and the modified E. coli trxB− /gor− (Origami) strain to overcome the problem of removing the GST moiety and to over-express and purify the rhIFN␣2b. Our results show the production of soluble and functional rhIFN␣2b at a yield of 100 mg/L, without optimization of any step of the process. The antiviral specific activity of the purified soluble IFN␣2b varied from 1.1 × 108 to ∼2.0 × 108 IU/mg as compared to the WHO IFN␣ standard. Our data show that production at high yield of a soluble and functional rhIFN␣2b can be achieved in E. coli when the protein is tagged with GST, and upon engineering of the GST-IFN junction this strategy can be applied to other proteins of medical interest. doi:10.1016/j.jbiotec.2007.07.123
S71
6. Cell stage-specific alpha-dystrobrevin distribution in subcellular compartments and association with other proteins in human cancer cells Veronika Borutinskaite a,∗ , Ruta Navakauskiene b , Karl-Eric Magnusson a a
Linkoping University, Veronika Borutinskaite, medicinsk mikrobiologi, Halsouniversitetet, Linkoping SE 581 85, Sweden b Institute of Biochemistry, Vilnius, Lithuania The dystrophin-associated protein complex (DPC) is expressed in muscle and non-muscle tissues. It is considered to be not only a mechanical component of cells, that links the cortical actin cytoskeleton to the extracellular matrix, but also a highly dynamic multifunctional structure that can serve as a scaffold for signaling proteins. Part of the signaling function of the DPC may be mediated by the dystrobrevins (DB). The involvement of DB in signaling pathways depends on the interaction with other proteins, like dystrophin, syntrophin, sarcoglycan complex and actin. In this study we have studied the possible role of alpha-DB in human non-muscle cancer cells (NB4 and HeLa). Analysing DB isoform expression in different cell fractions, total hydrophilic and hydrophobic proteins were isolated from proliferating NB4 cells as well as from cell nuclei only. We found that the alpha-DB isoforms were expressed differently in the NB4 cell fractions. We demonstrated that the hydrophilic fraction contained all major alpha-DB isoforms. In contrast, only alpha-DB-1 (77 kDa) was detected in the hydrophobic fraction of either cellular or nuclear membranes; this isoform could be important for cytoskeleton reorganisation during cell proliferation. Retinoids have been extensively studied over the years, and it has seen that all trans-retinoic acids (ATRA) likely provide the best example of the successful use in the treatment of acute promyelocytic leukemia (APL). Recently we have shown that combinations of ATRA with specific histone deacetylase inhibitors (HDACI) have synergistic effects on leukemia cell differentiation in vitro. Moreover, HDACI affect cancer cell division, induce differentiation and/or apoptosis (Savickiene et al., 2006). It has also been shown that alpha-DB undergoes phosphorylation which can be important in signal transduction (Grady et al., 2003). Using immunoprecipitation, we determined alphaDB phosphorylation after treatments with HDACI, BML-210 and ATRA. We found changes in tyrosine phosphorylation of alpha-DB-2 after treating NB4 cells with ATRA and BML210. In contrast, a combination of ATRA and BML-210 did not effected the phosphorylation level neither in the cytoplasmic nor in the nuclear fraction. With mass spectrometry analysis, new proteins interacting with alpha-DB were discovered. Confocal microscopy was used for visualization of alpha-DB localization in the cell, where colocalizations with actin and Hsp90 were established.