The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein.Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorpor.1te the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body. At least five proteins are essential for motor rotation (41, 47), but none of these have been found within the flagellar basal body (1,. 20), even though the basal body is considered a major part of the motor. Two of these proteins, MotA and MotB, are integral to the cell membrane (6, 37, 41). They are not necessary for assembly of the flagellum and do not copurify with it (20). They can be synthesized after flagellar assembly and used to activate the motor; paralyzed motA motB mutants acquire the ability to rotate their flagella after MotA and MotB synthesis under the direction of lambda-E. coli hybrid bacteriophage (41). Recently, it was shown (6) that, at least in the case of MotB, this acquisition of motility proceeds by quantum increases in flagellar rotation rate, presumably as a result of successive incorporation of subunits of MotB protein. The fact that motA and motB null mutants are nonetheless flagellated makes these genes different from all other flagellum-associated genes and will have * Corresponding author.

No abstract

Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova TM BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0-49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms.

We overexpressed the CheY protein by fusing the cheY gene to the tryptophan promoter from Serratia marcescens. Expression of the trp promoter-cheY fusion and subsequent purification of the protein resulted in the isolation of up to 20 mg of homogeneously pure CheY protein from 100 mg of the cytoplasmic supernatant fraction. Purification of the CheY protein was accomplished by exploiting the affinity of CheY protein to cibacron blue dye and molecular sieve chromatography. Preliminary biochemical characterization of the pure CheY protein revealed specific interactions with S-adenosylmethionine and cibacron blue dye. Additional kinetic analysis showed that CheY protein inhibits EcoRI methyltransferase. The amino acid composition of the CheY protein predicted by the DNA sequence of the cheY gene and the amino acid analysis of the CheY protein were in agreement, confirming the authenticity of the purified CheY protein.