Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.

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Cloning and Sequencing of a Poly(dl-Lactic Acid) Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13 and Its Functional Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2498-2504.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2498-2504 ◽

Cited By ~ 39

Author(s):

Yukie Akutsu-Shigeno ◽

Teerawat Teeraphatpornchai ◽

Kamonluck Teamtisong ◽

Nobuhiko Nomura ◽

Hiroo Uchiyama ◽

...

Keyword(s):

Escherichia Coli ◽

Lactic Acid ◽

Amino Acid ◽

Functional Expression ◽

Amino Acid Residues ◽

Cloning And Sequencing ◽

Gene Encoding ◽

Butylene Succinate ◽

Expression In Escherichia Coli ◽

Poly Ethylene

ABSTRACT The gene encoding a poly(dl-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(ε-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.

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Analysis of rare variations reveals roles of amino acid residues in the N-terminal extracellular domain of nicotinic acetylcholine receptor (nAChR) alpha6 subunit in the functional expression of human alpha6*-nAChRs

Molecular Brain ◽

10.1186/1756-6606-7-35 ◽

2014 ◽

Vol 7 (1) ◽

pp. 35 ◽

Cited By ~ 4

Author(s):

Bhagirathi Dash ◽

Ming D Li

Keyword(s):

Amino Acid ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Extracellular Domain ◽

Functional Expression ◽

Amino Acid Residues ◽

Nicotinic Acetylcholine

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The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1459 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1459-1464 ◽

Cited By ~ 115

Author(s):

Y Minami ◽

Y Kimura ◽

H Kawasaki ◽

K Suzuki ◽

I Yahara

Keyword(s):

Amino Acid ◽

Terminal Region ◽

Full Length ◽

Yeast Cells ◽

Amino Acid Residues ◽

Haploid Strain ◽

Carboxy Terminal Region ◽

Carboxy Terminal ◽

Hsp90 Alpha

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.

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Cloning, functional expression, and complementation analysis of an inorganic pyrophosphatase fromBartonella bacilliformis

Canadian Journal of Microbiology ◽

10.1139/m97-106 ◽

1997 ◽

Vol 43 (8) ◽

pp. 734-743 ◽

Cited By ~ 9

Author(s):

Samuel J. Mitchell ◽

Michael F. Minnick

Keyword(s):

Amino Acid ◽

Functional Expression ◽

Relative Activity ◽

Inorganic Pyrophosphatase ◽

Amino Acid Residues ◽

Bartonella Bacilliformis ◽

E Coli ◽

Catalytic Active Site

We have cloned the inorganic pyrophosphatase gene (ppa) from the facultative intracellular pathogen Bartonella bacilliformis and characterized its encoded product. The 531-bp gene is located approximately 1 kb downstream of, and in opposite orientation to, the invasion-associated locus (ialAB) of B. bacilliformis. The predicted protein encoded by ppa is 177 amino acid residues, which is in agreement with in vitro and in vivo synthesis of a protein with an apparent molecular mass of 22–23 kDa. The predicted B. bacilliformis pyrophosphatase (PPase) sequence is 53% identical and 85% similar to the E. coli PPase (EC 3.6.1.1), and contains all 12 of the amino acid residues implicated in the catalytic active site. The isolated B. bacilliformis PPase exhibits an activity of 51 ± 2 μmol PO4released/(mg protein∙min) at 28 °C and pH 8, and is sensitive to inhibition by Ca2+. In keeping with other prokaryotic PPases, B. bacilliformis PPase activity occurs from pH 6 to 10 (optimal pH = 8) and demonstrates high thermostability in the presence of Mg2+(highest activity at 55 °C, relative activity = 80 ± 3% at pH 8). The cloned B. bacilliformis ppa is able to genetically complement a ppa−mutant strain of E. coli.Key words: Bartonella, invasion-associated locus, inorganic pyrophosphatase.

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Molecular cloning and functional expression of a VIP-specific receptor

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00138.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. G728-G734 ◽

Cited By ~ 12

Author(s):

Huiping Zhou ◽

Jiean Huang ◽

Karnam S. Murthy

Keyword(s):

Smooth Muscle ◽

Amino Acid ◽

Functional Expression ◽

Amino Acid Residues ◽

Specific Receptor ◽

High Potency ◽

Functional Studies ◽

High Affinity ◽

Muscle Receptor ◽

Gastric Smooth Muscle

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC1, with high affinity for PACAP, and VPAC1 and VPAC2 with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIPs) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH2-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC2 receptor from GP gastric smooth muscle and VIPs from GP teniae coli smooth muscle. The cDNA sequence of the VIPs encodes a 437-amino acid protein ( Mr 49,560) that possesses 87% similarity to VPAC2 receptors in rat and mouse and differs from the VPAC2 receptor in GP gastric smooth muscle by only two amino-acid residues, F40F41 in lieu of L40L41. In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC50 1.4 nM) and stimulated cAMP formation with high potency (EC50 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC50 2.3 nM) and stimulated cAMP with equally high potency (EC50 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIPs distinct from VPAC1 and VPAC2 receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L40L41) in the NH2-terminal ligand-binding domain.

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Cloning, Characterization, and Functional Expression of Phospholipase Dα cDNA from Banana (Musa acuminate L.)

Journal of Food Quality ◽

10.1155/2017/2510949 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7

Author(s):

Li Li ◽

Xuemei He ◽

Jian Sun ◽

Changbao Li ◽

Dongning Ling ◽

...

Keyword(s):

Amino Acid ◽

Functional Expression ◽

Postharvest Quality ◽

Banana Fruit ◽

Amino Acid Residues ◽

Adaptive Responses ◽

Rt Pcr ◽

Reading Frame ◽

Flower Stem ◽

Different Temperatures

Phospholipase D (PLD) plays a key role in adaptive responses of postharvest fruits. A cDNA clone of banana (Musa acuminate L.) PLDα (MaPLDα) was obtained by RT-PCR in this study. The MaPLDα gene contains a complete open reading frame (ORF) encoding a 92-kDa protein composed of 832 amino acid residues and possesses a characteristic C2 domain and two catalytic H×K×××D (abbr. HKD) motifs. The two HKD motifs are separated by 341 amino acid residues in the primary structure. Relatively higher PLD activity and expression of MaPLDα mRNA were detected in developing tissues compared to senescent or mature tissues in individual leaves, flower, stem, and fruit organs, respectively. The expression profile of PLDα mRNA in postharvest banana fruits at different temperatures was determined, and the MaPLDα mRNA reached the highest expression peak on day 5 at 25°C and on day 7 at 12°C. The results provide useful information for maintaining postharvest quality and extending the storage life of banana fruit.

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Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors

International Journal of Molecular Sciences ◽

10.3390/ijms23010277 ◽

2021 ◽

Vol 23 (1) ◽

pp. 277

Author(s):

Yosuke Fukutani ◽

Yuko Nakamura ◽

Nonoko Muto ◽

Shunta Miyanaga ◽

Reina Kanemaki ◽

...

Keyword(s):

Amino Acid ◽

Hot Spot ◽

Olfactory Receptors ◽

Functional Expression ◽

Single Amino Acid ◽

Amino Acid Residues ◽

Membrane Expression ◽

Odor Molecule ◽

Molecule Recognition ◽

G Protein Coupled

Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs.

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Cloning, Overexpression, and Mutagenesis of theSporobolomyces salmonicolor AKU4429 Gene Encoding a New Aldehyde Reductase, Which Catalyzes the Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate to Ethyl (S)-4-Chloro-3-Hydroxybutanoate

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5207-5211.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5207-5211 ◽

Cited By ~ 25

Author(s):

Keiko Kita ◽

Takanobu Fukura ◽

Koh-Ichi Nakase ◽

Kenji Okamoto ◽

Hideshi Yanase ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Inhibition ◽

Amino Acid Sequences ◽

Yeast Cells ◽

Binding Motif ◽

Aldehyde Reductase ◽

Amino Acid Residues ◽

Gene Encoding ◽

E Coli

ABSTRACT We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolorAKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3β-hydroxysteroid dehydrogenase–plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303–2310, 1996). The ARII protein was overproduced inEscherichia coli about 2,000-fold compared to the production in the original yeast cells. The enzyme expressed inE. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G19-X-X-G22-X-X-A25, which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G19→A and G22→A mutant enzymes by 4-COBE did not occur. The A25→G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.

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