TY - JOUR AB - Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin1-90) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin1-90 assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin1-90. Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein. AU - Tan, H.L.* AU - Su, W.* AU - Wang, P.* AU - Zhang, W.* AU - Sattler, M. AU - Zou, P. C1 - 51723 C2 - 43465 CY - San Diego SP - 74-80 TI - Expression and purification of a difficult sarcomeric protein: Telethonin. JO - Protein Expr. Purif. VL - 140 PB - Academic Press Inc Elsevier Science PY - 2017 SN - 1046-5928 ER - TY - JOUR AB - Type II diabetes is characterized by deposition of the hormone human Islet Amyloid Polypeptide (hIAPP). Formation of hIAPP amyloid fibrils and aggregates is considered to be responsible for pancreatic β-cell losses. Therefore, insight into the structure of hIAPP in the solid-state and in solution is of fundamental importance in order to better understand the action of small molecules, which can potentially dissolve protein aggregates and modulate cell toxicity. So far, no procedure has been described that allows to obtain the native human IAPP peptide at high yields. We present here a cloning, expression and purification protocol that permits the production of 2.5 and 3 mg of native peptide per liter of minimal and LB medium, respectively. In the construct, hIAPP is fused to a chitin binding domain (CBD). The CBD is subsequently cleaved off making use of intein splicing reaction which yield amidation of the C-terminus. The N-terminus contains a solubilization domain which is cleaved by V8 protease, avoiding additional residues at the N-terminus. The correct formation of the disulfide bond is achieved by oxidation with H2O2. AU - Rodriguez Camargo, D.C. AU - Tripsianes, K.* AU - Kapp, T.G.* AU - Mendes, J. AU - Schubert, J. AU - Cordes, B.* AU - Reif, B. C1 - 42872 C2 - 35648 SP - 49-56 TI - Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli. JO - Protein Expr. Purif. VL - 106 PY - 2015 SN - 1046-5928 ER - TY - JOUR AB - Mycobacterium tuberculosis protein kinase G (PknG) is secreted into host macrophages to block lysosomal degradation. The catalytic domain (∼147-405) is C-terminally flanked by a tetratricopeptide repeat domain (TPRD). The preceding rubredoxin-like metal-binding motif (RD, ∼74 -147) mediates PknG redox regulation. The N-terminal ∼75 residues were predicted to show no regulatory secondary structure (NORS) and harbor the only site (T63) phosphorylated in vivo. Deletions or mutations in the NORS or the redox-sensitive RD significantly decrease the survival function. Here, we show that the RD appears only to be present in the folded, metal-bound state if ZnCl2 is added upon induction of protein expression in minimal medium. Since factor Xa cleaves at the end of its recognition site (IEGR), a modified expression plasmid for PknG 1-147 was obtained by mutating the N-terminal thrombin to a factor Xa recognition site. This allows preparing PknG1-147 with its native N-terminus. We further present a fast approach to generate expression plasmids for only the NORS or the RD by site-directed mutagenesis of the expression plasmid for His-tagged PknG 1-147. An expression plasmid for PknG 1-75 was obtained by introducing a stop codon at position 76 and one for PknG 74-174 by introducing a factor Xa recognition before position 74. SDS-PAGE analysis shows that all fragments are highly expressed in E. coli and can be purified to high purity. Thereby, the established preparation protocols pave the route for the NMR structural characterization of PknG regulation by its N-terminal regions, which is demonstrated by the recorded initial (1)H-(15)N-HSQC spectra. AU - Wittwer, M.* AU - Dames, S.A. C1 - 44107 C2 - 36746 CY - San Diego SP - 68-74 TI - Expression and purification of the natively disordered and redox sensitive metal binding regions of Mycobacterium tuberculosis protein kinase G. JO - Protein Expr. Purif. VL - 111 PB - Academic Press Inc Elsevier Science PY - 2015 SN - 1046-5928 ER - TY - JOUR AB - Epstein-Barr nuclear antigen 1 (EBNA1) is the essential Epstein-Barr virus (EBV) protein at the interface between the EBV genome and the host chromatin. It is EBNA1's task to guarantee replication and segregation of the multicopy closed circular viral genome in infected cells. While EBNA1's functions are relatively well understood, little is known about the molecular mechanisms of EBNA1 mediating chromatin tethering and DNA replication. To characterize those, purified EBNA1 would be a very useful tool in many different biochemical assays. For long, it was not possible to overexpress sufficient quantities of EBNA1 in Escherichia coli (E. coli) due to its rare codon usage, especially in the N-terminal part of the protein. Recently, some groups succeeded in purifying EBNA1 from bacteria using advanced inducible E. coli cells [1-3]. However, all purification procedures ended in a His-tagged version of EBNA1, which might influence EBNA1's function in biological assays. Therefore, we inserted a tobacco etch virus (TEV)-cleavage site between the N-terminal His-tag and the following open reading frame of EBNA1. Using sequential Ni-NTA and gel filtration columns and TEV protease-mediated cleavage upon autoinduction, we were able to purify functional EBNA1 protein featuring just a single additional, artificial N-terminal glycine residue. Following our simple and fast purification scheme we were able to synthesize 2mg of highly pure EBNA1 protein per liter culture. AU - Mayer, C.E. AU - Geerlof, A. AU - Schepers, A. C1 - 10801 C2 - 30368 SP - 7-11 TI - Efficient expression and purification of tag-free Epstein-Barr virus EBNA1 protein in Escherichia coli by auto-induction. JO - Protein Expr. Purif. VL - 86 IS - 1 PB - Elsevier PY - 2012 SN - 1046-5928 ER - TY - JOUR AB - Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni2+ affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 11 of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins. AU - Bogomolovas, J.* AU - Simon, B.* AU - Sattler, M. AU - Stier, G.* C1 - 451 C2 - 26082 SP - 16-23 TI - Screening of fusion partners for high yield expression and purification of bioactive viscotoxins. JO - Protein Expr. Purif. VL - 64 IS - 1 PB - Academic Press Inc Elsevier Science PY - 2009 SN - 1046-5928 ER - TY - JOUR AB - CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily, It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GSThCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NI M and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies. AU - Lechmann, M.* AU - Kremmer, E. AU - Sticht, H.* AU - Steinkasserer, A.* C1 - 9922 C2 - 20250 SP - 445-452 TI - Overexpression, Purification and Biochemical Characterization of the Extracellular Human CD83 Domain and Generation of Monoclonal Antibodies. JO - Protein Expr. Purif. VL - 24 PB - Elsevier PY - 2002 SN - 1046-5928 ER -