Beat Wipf

Two-dimensional map of the proteome of Haemophilus influenzae

We have constructed a two‐dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two‐dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low‐copy‐number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope‐bound protein fraction using either immobilized pH gradient strips or a two‐detergent system with a cationic detergent in the first and an anionic detergent in the second‐dimensional separation. Different proteins (502) were identified by matrix‐assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two‐dimensional proteome databases.

HMQC and HSQC experiments with water flip-back optimized for large proteins

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a 15N, 1H-HMQC spectrum of uniformly 15N/2H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different 15N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-15N-HMQC and 3D15 N-HMQC-NOESY-15N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly 15N/13C/2H-labelled S. aureus DNA gyrase B.

Non-GMP plasmid production for transient transfection in bioreactors

We describe a generic plasmid purification process for producing DNA for larger-scale transient transfection. Data on plasmid quality with regard to residual protein, endotoxin content and presence of different plasmid forms is given. The effects of contaminants and plasmid forms on expression levels of TNFRp55 and SEAP are discussed. Transient transfection of serum-free suspension grown mammalian cells represents a suitable approach to provide research quantities of proteins (50–100 mg) within1–2 weeks.

Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris

Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP-phosphoramidon binary complex and the apo enzyme.

Development and validation of a protocol for cell line identification by MALDI-TOF MS

Misidentification or cross-contamination of cell lines used in biotechnology or diagnostic settings are a challenge for laboratories and cell culture repositories. Masters et al. [1] among others reported the occurrence of large numbers of unrecognized and unreported misidentification or cross-contamination of cell lines. Current methods for the authentication of cell lines such as karyotyping, 2D-gel-electrophoresis, restriction fragment length polymorphism (RFLP) or short tandem repeats (STR) are expensive, labor intensive and not routinely applied. In the last decade MALDI-TOF MS became a powerful tool for the rapid and cost effective identification and taxonomic classification of microorganisms directly from whole cell extracts. We adapted this protein fingerprinting approach for a fast species identification of eukaryotic cell lines and established as well as validated a reference database. In addition we demonstrate that this new approach has a potential for the rapid characterization of recombinant protein expression systems. Accurate protein expression and the determination of the molecular mass of the recombinant expressed proteins (20-120kDa) can directly be analyzed from stable transfected and virus infected cultures.