High-level expression and characterization of a purified 142-residue polypeptide of the prion protein.

The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.

Chemotactic methyltransferase promotes adaptation to repellents in Bacillus subtilis.

Bacillus subtilis cheRB, which encodes the chemotactic methyltransferase, has been cloned and sequenced. CheRB is a polypeptide of 256 amino acids, with a predicted molecular mass of 28 kDa. A comparison of the predicted amino acid sequence of B. subtilis CheRB with that of Escherichia coli CheRE demonstrates that the two enzymes share 31% amino acid identity. The homology was functional in that the expression of cheBB in an E. coli cheRE null mutant made the bacteria Che+. In contrast to cheRE null mutants which show a strong smooth swimming bias, cheRB null mutants were predominantly tumbly. They respond to the addition and subsequent removal of attractant. They also respond to the addition of repellent but do not adapt; they resume prestimulus bias on removal of repellent. Tethering analysis of a culture of a cheRB null mutant revealed two distinct subpopulations, each demonstrating unique behaviors. One showed a strong clockwise flagellar rotation bias, whereas the other was more random. The latter phenotype may be due to a deficiency of CheB and may reflect an interaction of CheB and CheR. Measurements of CheB activity in the cheR null mutant showed them to be only 20% of wild type levels. We conclude from this work that CheRB functions to promote adaptation to repellent stimuli in B. subtilis, whereas CheRE functions to promote adaptation to attractant stimuli in E. coli.

Selection of an anti-IGF-1 Fab from a Fab phage library created by mutagenesis of multiple CDR loops.

Diverse Fab libraries containing 2-3 x 10(8) members were generated by randomizing amino acid residues within four of the six complementarity determining regions of a humanized version of an anti-HER-2 Ab (hu4D5). These libraries were subsequently displayed on the surface of the filamentous bacteriophage M13 and selected for binding to three proteins: CD4, insulin-like growth factor 1 (IGF-1), and tissue plasminogen activator. An Fab-bacteriophage was isolated that showed specific binding to IGF-1. The affinity of this Fab was determined to be 3.5 microM.

Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli.

A shotgun cloning of Bacillus subtilis DNA into pBR322 yielded a 2-kb fragment that suppresses the cold-sensitive defect of the nusA10(Cs) Escherichia coli mutant. The responsible gene encodes an open reading frame that is greater than 50% identical at the amino acid level to the E. coli rph gene, which was formerly called orfE. This B. subtilis gene is located at 251 degrees adjacent to the gerM gene on the B. subtilis genetic map. It has been named rph because, like its E. coli analog, it encodes a phosphate-dependent exoribonuclease activity, RNase PH, that removes the 3' nucleotides from precursor tRNAs. The cloned B. subtilis rph gene also suppresses the cold-sensitive phenotype of other unrelated cold-sensitive mutants of E. coli, but not the temperature-sensitive phenotype of three temperature-sensitive mutants, including the nusA11(Ts) mutant, that were tested.

Humanization of an anti-p185HER2 antibody for human cancer therapy.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

Fab assembly and enrichment in a monovalent phage display system.

We have developed a system that allows the expression of a single copy of an antibody Fab molecule on the surface of the filamentous bacteriophage M13 and demonstrate the utility of this system for enrichment of specific "Fab phage". A "humanized" version of antibody 4D5 (h4D5) directed against the extracellular domain of the HER2 (neu) receptor, was used as prototype to assess the assembly of Fab molecules on the phage and to determine the power of the enrichment system. The h4D5 Fab phage showed specific binding to the extracellular domain of the receptor and exhibited an affinity for its antigen virtually identical to the Fab itself. By virtue of its antigen binding, the h4D5 Fab phage could be sorted from a million-fold excess of non-cognate Fab phage in only two rounds of sorting. Further experiments demonstrated that the h4D5 Fab phage could be preferentially enriched from mixtures of variant Fab phages that had lower affinities for the HER2 receptor.

Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin.

The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.

An SfiI restriction map of the Bacillus subtilis 168 genome.

A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome. The combined sizes of the fragments indicate a genome size of approx. 4.2 Mb. The SfiI fragments range in size from 7-730 kb. Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map.

High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis.

The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.

The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis.

We have cloned and characterized the mtr operon of Bacillus subtilis. This operon encodes a presumed RNA-binding regulatory protein that is required for attenuation control of the trp operon. We have shown that the mtr operon consists of two structural genes, mtrA and mtrB, predicted to encode 22-kDa and 8-kDa polypeptides, respectively. MtrB shows homology with RegA, an RNA-binding regulatory protein of bacteriophage T4. The lesions in several mtr mutants were localized to mtrB or the putative mtr promoter. Several mtrB alleles were dominant to mtr+, suggesting that the regulatory factor is a multimeric protein. The in vivo action of the mtrA and mtrB gene products was analyzed in an E. coli strain containing a trpE-lacZ gene fusion under control of the B. subtilis trp promoter/attenuator region. Both MtrA and MtrB were necessary for regulation of beta-galactosidase production.

The promoter of the glucoamylase-encoding gene of Aspergillus niger functions in Ustilago maydis.

Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (HyR) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. HyR transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcriptional start points are the same in U. maydis and A. niger.

Engineering human prolactin to bind to the human growth hormone receptor.

A strategy of iterative site-directed mutagenesis and binding analysis was used to incorporate the receptor-binding determinants from human growth hormone (hGH) into the nonbinding homolog, human prolactin (hPRL). The complementary DNA for hPRL was cloned, expressed in Escherichia coli, and mutated to introduce sequentially those substitutions from hGH that were predicted by alanine-scanning mutagenesis and other studies to be most critical for binding to the hGH receptor from human liver. After seven rounds of site-specific mutagenesis, a variant of hPRL was obtained containing eight mutations with an association constant for the hGH receptor that was increased more than 10,000-fold. This hPRL variant binds one-sixth as strongly as wild-type hGH, but shares only 26 percent overall sequence identity with hGH. These studies show the feasibility of recruiting receptor-binding properties from distantly related and functionally divergent hormones and show that a detailed functional database can be used to guide the design of a protein-protein interface in the absence of direct structural information.

Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU.

The rates of synthesis of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis are controlled by a signal transduction pathway defined by at least four regulatory genes: degS, degU, degQ (formerly sacQ), and degR (formerly prtR). The DegS-DegU proteins show amino acid similarities with two-component procaryotic modulator-effector pairs such as NtrB-NtrC, CheA-CheY, and EnvZ-OmpR. By analogy with these systems, it is possible that DegS is a protein kinase which could catalyze the transfer of a phosphoryl moiety to DegU, which acts as a positive regulator. DegR and DegQ correspond to polypeptides of 60 and 46 amino acids, respectively, which also activate the synthesis of degradative enzymes. We show that the degS and degU genes are organized in an operon. The putative sigma A promoter of the operon was mapped upstream from degS. Mutations in degS and degU were characterized at the molecular level, and their effects on transformability and cell motility were studied. The expression of degQ was shown to be subject both to catabolite repression and DegS-DegU-mediated control, allowing an increase in the rate of synthesis of degQ under conditions of nitrogen starvation. These results are consistent with the hypothesis that this control system responds to an environmental signal such as limitations of nitrogen, carbon, or phosphate sources.

Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems.

Mutations in the sacU region have a pleiotropic phenotype. Certain mutations designated sacU(Hy), for example, express degradative enzymes at high levels, are able to sporulate in the presence of glucose, have severely reduced transformation efficiencies, and are nonmotile. We isolated and sequenced the sacU gene region of Bacillus subtilis. Two open reading frames were found in the sacU region, and sacU(Hy) mutations were localized to both of these open reading frames. The two open reading frames have similarities to two widespread families of proteins that mediate responses to environmental stimuli.