The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of Paracoccus denitrificans was identified, and its corresponding gene was cloned and analyzed at the molecular level. The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (phaC
Pd) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774–779, 1996). Gene walking around phaC
Pdrevealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the phaP
Pd gene, encoding GA16 protein, and the other was thephaR
Pd gene, encoding a protein that is putatively involved in the regulation of the expression ofphaP
Pd. Overproduction of PhaPPdwas observed in Escherichia coli carryingphaP
Pd, but the overproduction was not observed in the presence of phaR
Pd. Coexpression ofphaP
Pd and PHA biosynthesis genes in E. coli caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB. GA16 protein was considered a phasin protein. ThephaR
Pd gene had significant similarities tostdC, a possible transcriptional factor of Comamonas testosteroni, as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria.
The heavy chain (Hc) and light chain (Lc) genes of the Fab fragment of a catalytic antibody 6D9 were simultaneously expressed in an Escherichia coli in vitro transcription/translation system without a reducing agent. The intermolecular disulfide bond between the Hc and Lc was found formed, suggesting a correct formation of the Fab fragment in the in vitro system. In enzyme-linked immunosorbent assay, the Fab fragment synthesized in vitro exhibited an antigen-binding activity. Addition of reduced glutathione, oxidized glutathione, protein disulfideisomerase and molecular chaperones, GroEL and GroES, increased the solubility and the antigen-binding activity of the Fab fragment greatly. The in vitro synthesized Fab was purified by means of a hexa-histidine tag attached to the C-terminus of the Hc. Catalytic assay of the purified Fab fragment showed that the His-tagged Fab fragment synthesized in vitro had a catalytic activity comparable to that produced in vivo. ß
A functional expression system for a heme protein of Phanerochaete chrysosporium, manganese peroxidase (MnP), was developed using the Escherichia coli in vitro coupled transcription/translation system in the presence of hemin and fungal protein disulfide isomerase. This system has allowed the high-throughput construction and screening of a large diversity of mutant heme enzymes and has made it possible to improve the enzymatic function efficiently. Here we increased the H2O2 stability of MnP using this system; a mutant MnP library containing three randomized amino acid residues located in the H2O2-binding pocket of MnP was designed and constructed on a 384-well plate using SIMPLEX (single-molecule-PCR-linked in vitro expression). The screening of 10(4) samples independently expressed for improved H2O2 stability led to four positive mutants, the H2O2 stability of which was nine times higher than that of the wild-type.
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