This invention is in the field of molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding arthropod protein disulfide isomerases.
Protein folding requires the assistance of folding helpers in vivo. The formation or isomerization of disulfide bonds in proteins is a slow process requiring catalysis. In nascent polypeptide chains the cysteine residues are in the thiol form. The formation of the disulfide bonds usually occurs simultaneously with the folding of the polypeptide, in the endoplasmic reticulum of eukaryotes or in the periplasm of Gram-negative bacteria. Cells contain three types of accessory proteins that function to assist polypeptides in folding to their native conformations: protein disulfide isomerases, propyl cis-trans isomerases, and molecular chaperones.
Protein disulfide isomerase (PDI) is a homodimeric eukaryotic enzyme which catalyzes disulfide interchange reactions. PDI is also thought to be the beta subunit of the heterotetramer prolyl hydrolase, the enzyme that hydroxylates the proline residues in Collagen. PDI appears to belong to a family of closely related proteins which have specific functions. PDI (EC 5.3.4.1), also called S-S rearrangase, catalyzes the rearrangement of both intrachain and interchain disulfide bonds in proteins to form native structures. The reaction depends on sulfhydryldisulfide interchange, and PDI needs reducing agents or partly-reduced enzyme. A family of PDI-like proteins have been identified in mammals, yeasts, fungi, plants, and Drosophila.
In Drosophila, a PDI precursor was identified by screening a genomic DNA library at reduced stringency hybridization conditions using a rat Phospholipase C alpha cDNA probe. Northern analysis showed that this gene encodes a transcript that is present throughout development, in heads and bodies of adults. The encoded protein contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a C-terminal ER-retention signal (KDEL). Overall, this Drosophila PDI gene contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat Phospholipase C alpha (30% identical) (McKay et al. (1995) Insect Biochem Mol Biol 25:647-654).
Another member of the PDI family is ERp-60, a PDI isoform initially misidentified as a phosphatidylinositol-specific phospholipase C. The human and Drosophila ERp60 polypeptides have been cloned and expressed. These two ERp-60 polypeptides are similar to human PDI within almost all their domains, the only exception being the extreme C-terminal region. Coexpression in insect cells of the human or Drosophila ERp-60 with the alpha subunit of human propyl 4-hydrolase does not result in tetramer formation or prolyl 4-hydroxylase activity in the cells. This lack of tetramer formation is not only due to the differences in the C-terminal region since no prolyl 4-hydroxylase tetramer is formed when a human ERp-60 hybrid containing the C-terminal region of the human PDI polypeptide is used (Koivunen et al. (1996) Biochem J 316:599-605). The 5xe2x80x2 flanking region of the ERp-60 gene has no TATAA box or CCAAT motif but contains several potential binding sites for transcription factors. The highest levels of expression of the human ERp-60 mRNA are found in the liver, placenta, lung, pancreas, and kidney, and the lowest in the heart, skeletal muscle, and brain. The ERp-60 gene has been mapped by fluorescence in situ hybridization to 15q15, a different chromosome than where the human PDI and thioredoxin genes are found (Koivunen, et al. (1997) Genomics 42:397-404).
Full-length cDNA clones encoding two members of the mice PDI family have been cloned, sequenced, and expressed (ERp-59/PDI and ERp-72). ERp-59/PDI has been identified as the microsomal PDI. The ERp-72 amino acid sequence shares sequence identity with ERp-59/PDI at three discrete regions, having three copies of the sequences that are thought to be the CGHC-containing active sites of ERp-59/PDI. ERp-59/PDI has the sequence KDEL at its COOH terminus while ERp72 has the related sequence KEEL (Mazarella et al. (1990) J Biol Chem 265:1094-1101). A cDNA clone containing sequence similarity to the mammalian lumenal endoplasmic reticulum protein ERp-72 has been isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA encoding human PDI. The polypeptide encoded by this cDNA possesses a putative N-terminal secretory signal sequence and two regions identical to the active sites of PDI and ERp-72. This protein appears to be encoded by a small gene family in alfalfa, whose transcripts are constitutively expressed in all major organs of the plant. In alfalfa cell suspension cultures, ERp-72 transcripts are induced by treatment with tunicamycin, but not in response to calcium ionophore, heat shock or flingal elicitor (Shorrosh and Dixon (1992) Plant J 2:51-58)
Another member of the PDI family is ERp-5. The amino acid sequence deduced from its cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence are found in the active sites of rat and human PDI and in Form I phosphoinositide-specific phospholipase C. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with ERp-5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells (Chaudhuri et al. (1992) Biochem J 281:645-650).
A PDI-like protein from Acanthamoeba castellanii contains two highly conserved thioredeoxin-like domains, each about 100 amino acids. However, the A. castellanji PDI-like protein differs from other members in many aspects, including the overall organization and isoelectric point. Southern and Northern analyses demonstrate that the PDI-like protein is encoded by a single-copy gene which is transcribed to generate a 1500-nucleotide mRNA (Wong and Bateman (1994) Gene 150:175-179).
Included in this application are scorpion, spider, lepidoptera, and centepede ESTs with similarities to several of these PDIs. Coexpression in plants or insect cells of an arthropod PDI with a secreted arthropod protein should enhance the yield of the foreign protein by increasing the proper folding of the foreign protein.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a protein disulfide isomerase precursor polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a spider, a centipede, a moth, and a scorpion protein disulfide isomerase of SEQ ID NOs:2, 4, 6, and 8. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding an ERp60 polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a spider, a centipede, and a scorpion ERp60 of SEQ ID NOs:10, 12, and 14. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding an ERp72 polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a two scorpion ERp72s of SEQ ID NOs:16 and 18. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding an ERp5 polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a moth, a worm, and two scorpion ERp5s of SEQ ID NOs:20, 22, 24, and 26. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotide of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15,17,19, 21, 23, and 25 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12,14, 16,18, 20, 22, 24, and 26. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11,13,15,17,19, 21, 23, 25 and the complement of such nucleotideqsequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide (such as ERp-60, an ERp-72, an ERp-5, or a PDI-like homolog) of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be of eukaryotic origin, such as an insect, a yeast, or a plant cell; of prokaryotic origin, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a protein disulfide isomerase precursor polypeptide of at least 30 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, and 8. The present invention relates to an ERp60 polypeptide of at least 40 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:10, 12, and 14. The present invention relates to an ERp72 polypeptide of at least 20 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:16 and 18. The present invention relates to an ERp5 polypeptide of at least 50 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:20, 22, 24, and 26.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a protein disulfide isomerase polypeptide in a host cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a plant cell;
measuring the level an enzyme polypeptide in the plant cell containing the isolated polynucleotide; and
comparing the level of an enzyme polypeptide in the plant cell containing the isolated polynucleotide or an isolated chimeric gene with the level of an enzyme polypeptide in a plant cell that does not contain the isolated polynucleotide or an isolated chimeric gene.
presentinvention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a protein disulfide isomerase polypeptide gene, preferably an arthropod polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a protein disulfide isomerase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a protein disulfide isomerase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
The instant invention relates to isolated nucleic acid fragments encoding arthropod protein disulfide isomerases. Specifically, this invention concerns an isolated nucleic acid fragment encoding an ERp-60, an ERp-72, an ERp-5, or a PDI-like homolog. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding ERp-60, ERp-72, ERp-5, or PDI-like homolog.
Another embodiment of the instant invention pertains to a method for expressing a gene encoding a protein disulfide isomearase in the genome of a recombinant baculovirus in insect cell culture or in viable insects wherein said insect cells or insects have been genetically engineered to express an ERp-60, an ERp-72, an ERp-5, or a PDI-like homolog.