Ilkka Palva

Hormone-sensitive lipase is closely related to several bacterial proteins, and distantly related to acetylcholinesterase and lipoprotein lipase: Identification of a superfamily of esterases and lipases

We have sequenced a gene from Bacillus acidocaldarius which encodes an open reading frame (ORF3) of 310 amino acids. The ORF3 was found to be related to the mammalian hormone-sensitive lipase (HSL). Searching the protein data base revealed five other bacterial proteins related to the HSL. Upon further sequence comparisons this HSL-group was found to be related to the family of carboxylesterases, and to a family of lipases (lipoprotein, hepatic and pancreatic lipases). The evolutionary relationship of these serine-dependent hydrolytic enzymes has not been studied previously, and it has not been known that these proteins belong to the same superfamily. Finally, the alignment of the HSL with the bacterial proteins allowed us to infer the location of the hormone-sensitive regulatory domain of the HSL-protein.

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis.

SummaryThe gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the α-amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-polyacrylamide gel electrophoresis characterizations. The NH2-terminal sequence analysis of the secreted PME revealed two different NH2-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and α-amylase signal peptide.

Expression of the outer membrane protein P.69 ofBordetella pertussis inBacillus subtilis

SummaryThe gene coding forBordetella pertussis P.69 protein was cloned and expressed inBacillus subtilis. The expression vector contained the promoter region and the sequence coding for the whole or truncated signal sequence of the α-amylase gene fromB. amyloliquefaciens. Using either construction the level of expression was relatively low and the protein was found in the particulate fraction. The protein migrated in gel electrophoresis slower than expected from its deduced amino acid content thereby giving the appearance of having an anomalously large molecular mass.

Bacillus—a Promising Tool for Genetic Engineering

РЕЗЮМЕНесмотря на то, что Е. coli продолжает оставаться самым распространенным объектом в роли продуцента чужеродных белков, интенсивно изучаются и альтернативные системы клеток-хозяев экзогенной ДИК. Продуцированием белков с их последующей секрецией клетками-хозяевами можно преодолеть многие из проблем, с которыми сталкиваются при использовании системы Е. coli. Чтобы применять секрецию, необходимо знать ее молекулярную основу. В статье дан краткий обзор существующих теорий по данной проблеме.Попытка авторов использовать альтернативную систему клеток-хозяев была направлена на интенсивное изучение Bacillus. Экзоэнзим α-амилаза из B.amyloliquefaсiens был впервые клонирован в мультикопийной плазмиде и успешно экспрессирован и секретирован в B.subtilis. Сконструирован вектор для секреции на основе регуляторных сигналов гена α-амилазы и изучено его функционирование в сочетании с различными модельными генами. Изучены также подходы к совершенствованию системы клеток-хозяев. Перспективной возможностью усилить про...