Hans-Werner Lahm

Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor

Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.

Hydrolysis and amino acid composition analysis of proteins

Amino acid composition analysis is a classical protein analysis method, which finds a wide application in medical and food science research and is indispensable for protein quantification. It is a complex technique, comprising two steps, hydrolysis of the substrate and chromatographic separation and detection of the residues. A properly performed hydrolysis is a prerequisite of a successful analysis. The most significant developments of the technology in the last decade consist in the (i) reduction of the hydrolysis time by the use of microwave radiation energy; (ii) improvement in the sensitivity of the residue detection, the quantification of the sensitive residues and separation of the enantiomeric forms of the amino acids; (iii) application of amino acid analysis in the large-scale protein identification by database search; and (iv) gradual replacement of the original ion exchange residue separation by reversed-phase high-performance liquid chromatography. Amino acid analysis is currently facing an enormous competition in the determination of the identity of proteins and amino acid homologs by the essentially faster mass spectrometry techniques. The amino acid analysis technology needs further simplification and automation of the hydrolysis, chromatography and detection steps to withstand the pressure exerted by the other technologies.

TWO HUMAN TNF RECEPTORS HAVE SIMILAR EXTRACELLULAR, BUT DISTINCT INTRACELLULAR, DOMAIN SEQUENCES

Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.

Mass spectrometry : A tool for the identification of proteins separated by gels

Mass spectrometry (MS) has become the technique of choice to identify proteins. This has been largely accomplished by the combination of high‐resolution two‐dimensional (2‐D) gel separation with robotic sample preparation, automated MS measurement, data analysis, and database query. Developments during the last five years in MS associated with protein gel separation are reviewed.

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.

Characterization of recombinant human interleukin-2 with micromethods

Highly purified recombinant human interleukin-2, expressed in Escherichia coli, was analyzed by micromethods. N-Terminal sequence analysis showed that methionine at position 0 was found in 90% of the molecules and not completely removed in post-ribosomal processing. A complete peptide map of the reduced and S-carboxymethylated protein was obtained by high-performance liquid chromatography after tryptic digestion, and the fragments were identified by amino acid analysis and automated Edman sequence analysis. Using a double-label S-carboxymethylation procedure, it was determined that there is a disulfide linkage between the cysteine residues at positions 58 and 105. The third cysteine residue at position 125 was found to be present as the free sulfhydryl.

Characterisation and sequence of a protective rhoptry antigen from Plasmodium falciparum.

We have recently demonstrated that a non-polymorphic rhoptry antigen, RAP-1 (rhoptry associated protein-1), which is recognised by human immune serum, can successfully protect Saimiri monkeys from a lethal infection of Plasmodium falciparum malaria. In this report we further characterise the antigen, which consists of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa, and present the antigens gene sequence. Monoclonal antibody evidence, autocatalytic processing and immunological cross-reactivity suggest that all components of this antigen are derived from the same precursor protein. The antigen is lipophilic, and disulphide bonding plays an important role in its structure. We discuss the structure and function of RAP-1 in the light of its deduced amino acid sequence and consider the relationship of this antigen to other rhoptry antigens of similar subunit size and composition.

Effect of the hydrolysis method on the determination of the amino acid composition of proteins

Fast and reproducible separation and determination of amino acids serves the economical and reliable characterization and quantification of peptides and proteins as well as the identification of proteins by amino acid composition analysis on a large-scale. A prerequisite of a successful compositional analysis is a complete hydrolysis of the peptides and proteins and a quantitative recovery of the residues in the hydrolyzate. We investigated the effect of different acid-hydrolysis methods on the compositional analysis of known proteins in solution and after blotting onto polyvinylidene difluoride membranes and worked out the conditions for the processing of large numbers of samples. The reliability of each method was studied by introducing the analysis data into the AACompIdent software and deducing the protein identification scores. All acid-hydrolysis methods delivered reliable analysis data. The most accurate data were provided by conventional, thermal hydrolysis of proteins in solution in the presence of methanesulfonic acid, closely followed by hydrolysis with hydrochloric acid and microwave radiation-dependent hydrolysis with hydrochloric or methanesulfonic acid, respectively. For blotted proteins, conventional hydrolysis delivered more accurate analysis data in comparison with the microwave radiation-induced hydrolysis. The extraction of the residues from the membrane hydrolyzate was a critical step for unambiguous protein identification. Microwave radiation-induced hydrolysis was responsible for a higher degree of racemization of the residues.

Identification of a human immunodeficiency virus-1 protease cleavage site within the 66,000 dalton subunit of reverse transcriptase

The human immunodeficiency virus-1 reverse transcriptase is a heterodimer of related 51 and 66 kDa subunits. The smaller subunit arises by viral protease-catalyzed cleavage of the carboxy-terminal domain of the 66 kDa species. Comparison of the amino acid composition analyses of the isolated 51 kDa and 66 kDa subunits indicates that the carboxyl terminus of 51 kDa is Phe440. This site was confirmed in vitro using purified recombinant protease and a peptide spanning the postulated cleavage area. The sequence surrounding this site does not show significant homology to other protease cleavage sites in the viral gag and pol precursors; thus, this new information may contribute to our understanding of the sequence specificity of the viral protease.

The primary structure of monkey pituitary growth hormone.

Monkey growth hormone (MGH) has been purified by reverse-phase high-performance liquid chromatography (HPLC). Tryptic digests of MGH were separated by HPLC and paper electrophoresis. From amino acid composition, from NH2-terminal residue and sequence analyses of these tryptic peptides, and from their alignment with those of human growth hormone, the primary structure of MGH was proposed. There are only four residues which are different in these two growth hormone molecules.