High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Expression of beta 2-adrenoceptors in halobacteria.

Halobacteria are halophilic representatives of the recently defined domain, the Archaea. Halobacterium salinarium belongs to this group of microorganisms and contains large amounts of bacteriorhodopsin in its membrane. Bacteriorhodopsin is a seven-transmembrane protein that consists of bacterio-opsin (BO), and the chromophore retinal, which is covalently attached to BO. We have investigated whether the expression machinery for BO can be utilized for synthesis of the human beta 2-adrenoceptor (beta 2-AR), a protein with a similar seven-transmembrane-helix topology. An expression vector for BO synthesis was modified to express beta 2-ARs under the control of BO regulatory clements in H. salinarium. Homologous recombination into the genome was verified by polymerase chain reactions. Northern blots revealed transcripts of the calculated size and significant amounts of epitope-tagged beta 2-ARs were detected in Western blots. However, binding of the beta-AR antagonist 125I-cyanopindolol revealed low levels of functional receptors, and the ligand binding properties of these receptors were altered when compared to native receptors. Expression of chimeras containing larger amino terminal portions of BO did not result in higher receptor levels. Expression of beta 2-AR in Haloferax volcanii, another member of halobacteria, was achieved with a vector carrying the ferredoxin promoter. The levels of functional receptor as determined by 125I-cyanopindolol binding were 180 fmol/mg protein. The beta-AR ligands isoprenaline and propranolol showed affinities expected for functional beta 2-ARs. Thus, functional human beta 2-ARs were expressed in halobacteria, constituting a first approach for expression of a eukaryotic protein in the domain of Archaea.

Constitutively active germline mutation of the thyrotropin receptor gene as a cause of congenital hyperthyroidism.

Congenital hyperthyroidism is a rare, transient disease usually caused by transmission of thyrotropin receptor autoantibodies from the mother with Graves' disease to her child. We report a German women and her two sons who had congenital, but persistent hyperthyroidism without any signs of autoimmunity. Direct sequencing of the polymerase chain reaction-amplified exon 10 of the thyrotropin receptor genomic DNA revealed in the mother and both sons a transition of GCC to GTC, resulting in an exchange of alanine 623 to valine. This germline mutation in a highly conserved region of the thyrotropin receptor resulted in a constitutive activation of the cyclic adenosine monophosphate-generating cascade with resulting hyperthyroidism. Analysis of the family for a corresponding BstXI restriction-site polymorphism revealed heterozygosity for this mutation in the affected family members, but not in the father or other relatives. We conclude that whenever congenital hyperthyroidism is persistent and parameters of autoimmunity are absent, a constitutively active thyrotropin receptor mutation should be considered. Treatment appears to require aggressive means such as total thyroidectomy or ablation by 131iodine because two subtotal thyroidectomies in the mother were insufficient to control the disease.

Mutations of the TSH receptor as cause of congenital hyperthyroidism.

4 patients of two families with congenital persistent hyperthyroidism without detectable autoantibodies are reported. The members of the first family affected by hyperthyroidism, i.e. the mother and her two children, showed a germline mutation, a transition of GCC to GTC in the genomic DNA of the TSH receptor, leading to an exchange of alanine by valine at the position 623. The mother was thyroidectomized at two times because of recurrent nodular goiter. The third child of a healthy second family showed a transition of AGC to AAC leading to an exchange of serine by asparagine at the position 505 of the TSH receptor. The mutation of family 1, as a somatic point mutation leading to autonomous thyroid adenoma, has originally been demonstrated to constitutively activate TSH independent cAMP accumulation. The functional tests of the TSH receptor gen mutation, detected in family 2, are ongoing, but an exchange of serine by arginine at the same position has been shown to lead to constitutively active cAMP accumulation. The cases of congenital hyperthyroidism in the first family lead to a reduction of the birth weight and head circumference and to a neonatal but not fetal tachycardia. Bone age of both children was accelerated by one year. In contrast to that, congenital hyperthyroidism of the second family lead to more marked signs of intrauterine hyperthyroidism. The mother observed marked symptoms of fetal and neonatal hyperthyroidism. The bone age at a chronological age of 6 months was 4-6 years and the neonate showed a mild exophthalmus. We conclude, that congenital hyperthyroidism due to constitutively activating TSH receptor mutations has to be considered, if hyperthyroidism is not transient but persistent, and the parameters of autoimmunity are absent. Constitutively active TSH receptor germline mutations lead to different degrees of congenital hyperthyroidism. In contrast to patients with Graves' disease, more aggressive means of treatment like total thyroidectomy and/or radiation seem to be recommendable in cases with severe hyperthyroidism to control the disease.

Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors.

beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide-binding proteins (G-protein) subsequent to receptor phosphorylation by the beta-adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors.

Phosducin inhibits receptor phosphorylation by the beta-adrenergic receptor kinase in a PKA-regulated manner.

Homologous or receptor-specific desensitization of beta-adrenergic receptors is thought to be triggered by receptor phosphorylation mediated by the beta-adrenergic receptor kinases (beta ARK). Upon receptor activation, cytosolic beta ARK translocates to the membrane, probably by binding to G-protein beta gamma-subunits. Using the purified proteins reconstituted into phospholipid vesicles we show here that this binding process can be inhibited by phosducin, a cytosolic protein that has recently been described as a regulator of G-protein-mediated signalling. Phosducin appears to complete very effectively with beta ARK for the G-protein beta gamma-subunits. These inhibitory effects of phosducin on receptor phosphorylation are antagonized following phosphorylation of phosducin by protein kinase A. It is proposed that phosducin may act as a regulator of homologous beta-adrenergic receptor desensitization.

Purification and functional characterization of beta-adrenergic receptor kinase expressed in insect cells.

The beta-adrenergic receptor kinase mediates agonist-dependent phosphorylation of beta-adrenergic receptors, which is thought to represent the first step of homologous desensitization. We have expressed bovine and human beta ARK1 in Sf9 cells and purified them to apparent homogeneity in milligram quantities. The Km-values of the enzyme were 3.8 microM for rhodopsin and 22 microM for ATP; the Vmax-value was 9.9 mol phosphate/mol beta ARK/min. These data indicate that the two recombinant kinases were at least as active as preparations previously obtained from bovine brain. There were no differences in the functional activity of human and bovine beta ARK.

Expression in Escherichia coli of the genes coding for reaction center subunits from Rhodobacter sphaeroides: wild-type proteins and fusion proteins containing one or four truncated domains from Staphylococcus aureus protein A at the carboxy-terminus.

Gene cassettes were constructed containing Rhodobacter sphaeroides puhA, pufM and pufL sequences with synthetic 5' ends for production in Escherichia coli of the H, M and L subunits of the photosynthetic reaction center. In addition, gene cassettes coding for fusion proteins with proteinase recognition site(s) between the amino-terminal part of H, M or L subunits, and the carboxy-terminal part consisting of one (B') or four (D'ABC') domains of Staphylococcus aureus protein A were constructed. A modified expression vector pDS12/RBSII containing the T5 promoter PN25, the lac operator, and a newly inserted E. coli lipoprotein ribosome-binding site was used. Inducible synthesis of plasmid-encoded polypeptides was accompanied by reduced growth. The products comigrated with R. sphaeroides reaction center subunits H, M and L. They were identified by Western blot experiments using antibodies raised against reaction center proteins. The hybrid protein containing the reaction center H subunit fused to the single domain B' was not detected by nonspecific antisera. In contrast, the three fusion proteins containing domains D'ABC' were identified using nonspecific antisera. This indicated that domains D'ABC' were sufficient to bind to the Fc part of IgG molecules, whereas domain B' was not sufficient. This property was used to purify all three fusion proteins with domains D'ABC' by affinity chromatography from the membrane fraction of E. coli cells.