scholarly journals Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

Processes ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 2118
Author(s):  
Carina Bannach ◽  
Daniel Ruiz Buck ◽  
Genna Bobby ◽  
Leo P. Graves ◽  
Sainan Li ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Gene Promoter ◽  
Protein Product ◽  
Expression Vectors ◽  
Coding Region ◽  
Base Pairs ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Transfer Vectors

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.

scholarly journals Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi.

1984 ◽  
Vol 4 (6) ◽  
pp. 1115-1124 ◽  
Author(s):  
K J Brunke ◽  
J G Anthony ◽  
E J Sternberg ◽  
D P Weeks
Keyword(s):  
Transcription Initiation ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Amino Acid Sequences ◽  
Sequencing Data ◽  
Base Pairs ◽  
Tubulin Gene ◽  
Gene Set ◽  
Tubulin Genes ◽  
Beta 2

The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.

Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe

1990 ◽  
Vol 10 (7) ◽  
pp. 3750-3760
Author(s):  
P Sunnerhagen ◽  
B L Seaton ◽  
A Nasim ◽  
S Subramani
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Protein Product ◽  
Start Codon ◽  
Insertion Mutagenesis ◽  
Amino Acid Residues ◽  
Flanking Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
Repair Gene

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.

scholarly journals Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants.

1993 ◽  
Vol 13 (10) ◽  
pp. 6403-6415 ◽  
Author(s):  
S Connelly ◽  
W Filipowicz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Nicotiana Plumbaginifolia ◽  
Gene Promoters ◽  
Coding Region ◽  
Pol Ii ◽  
U2 Snrna ◽  
Snrna Gene

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

scholarly journals The Alternative Noncoding Exons 1 of Aromatase (Cyp19) Gene Modulate Gene Expression in a Posttranscriptional Manner

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3301-3307 ◽  
Author(s):  
Hanzhou Wang ◽  
Rong Li ◽  
Yanfen Hu
Keyword(s):  
Gene Expression ◽  
Posttranscriptional Regulation ◽  
Rna Stability ◽  
Postmenopausal Breast Cancer ◽  
Protein Translation ◽  
Protein Product ◽  
Expression Vectors ◽  
Coding Region ◽  
Tissue Specific ◽  
Exon 1

Aromatase (Cyp19) is a key enzyme in estrogen biosynthesis and an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. Aromatase transcription is driven by multiple tissue-specific promoters, which result in the production of various mRNA transcripts that contain an alternative noncoding exon 1 followed by a common protein-coding region. Transcriptional activity of these promoters is the only known determinant for aromatase protein abundance in a given tissue or cellular context. To determine whether aromatase expression could be influenced by additional regulatory mechanisms, we used a common heterologous promoter to drive the expression of multiple aromatase cDNA sequences that differ only by the alternative exon 1 sequence. These expression vectors gave rise to vastly different levels of aromatase mRNA and protein in multiple cell lines examined. Furthermore, the relative abundance of several mRNA variants did not correlate with that of the corresponding protein product. The variation in mRNA and protein levels is most likely due to a negative effect of certain alternative exons 1 on RNA stability and protein translation. Deletional analyses indicate that the 5′ regions of the adipose tissue-specific exons I.3 and I.4 contain the cis-acting elements responsible for modulation of aromatase levels. Thus, our work uncovers an important role of the alternative exons 1 in posttranscriptional regulation of aromatase gene expression.

scholarly journals Deletion of the Autographa californica nucleopolyhedrovirus chitinase KDEL motif and in vitro and in vivo analysis of the modified virus

2004 ◽  
Vol 85 (4) ◽  
pp. 821-831 ◽  
Author(s):  
Giles P. Saville ◽  
Alexandra L. Patmanidi ◽  
Robert D. Possee ◽  
Linda A. King
Keyword(s):  
Trichoplusia Ni ◽  
Lethal Dose ◽  
Gene Promoter ◽  
Autographa Californica ◽  
Coding Region ◽  
Insect Larvae ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Infected Cells

Infection of insect larvae with Autographa californica nucleopolyhedrovirus (AcMNPV) results in the liquefaction of the host, a process involving the action of virus-encoded chitinase and cathepsin gene products. Chitinase is localized in the endoplasmic reticulum (ER) during infection because of the presence of a C-terminal ER retrieval motif (KDEL). In this study, the KDEL coding region was removed from the chitinase gene so that expression of the modified chitinase remained under the control of its own gene promoter, at its native locus. The deletion of KDEL resulted in the redistribution of chitinase within the cell during virus infection. Chitinase lacking the KDEL motif was detectable at the plasma membrane and was also evident in the culture medium of virus-infected cells from as early as 12 h post-infection (p.i.). Secretion of chitinase from the cell continued up to 72 h p.i., until cytolysis. The biological activity of the recombinant virus in Trichoplusia ni larvae was enhanced, with a significant reduction in the lethal dose and lethal time associated with infection. Furthermore, a reduction in feeding damage caused by infected larvae was observed compared to AcMNPV-infected individuals.

scholarly journals Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe.

1990 ◽  
Vol 10 (7) ◽  
pp. 3750-3760 ◽  
Author(s):  
P Sunnerhagen ◽  
B L Seaton ◽  
A Nasim ◽  
S Subramani
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Protein Product ◽  
Start Codon ◽  
Insertion Mutagenesis ◽  
Amino Acid Residues ◽  
Flanking Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
Repair Gene

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.

Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants

1993 ◽  
Vol 13 (10) ◽  
pp. 6403-6415
Author(s):  
S Connelly ◽  
W Filipowicz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Nicotiana Plumbaginifolia ◽  
Gene Promoters ◽  
Coding Region ◽  
Pol Ii ◽  
U2 Snrna ◽  
Snrna Gene

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

Enhancer and silencerlike sites within the transcribed portion of a Ty2 transposable element of Saccharomyces cerevisiae

1989 ◽  
Vol 9 (11) ◽  
pp. 4824-4834
Author(s):  
P Farabaugh ◽  
X B Liao ◽  
M Belcourt ◽  
H Zhao ◽  
J Kapakos ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Fold Increase ◽  
Initiation Site ◽  
Heterologous Gene ◽  
Coding Region ◽  
Base Pairs ◽  
Steady State Levels ◽  
Rna Accumulation ◽  
A Site

The Ty2-917 element is a member of the Ty2 class of retroviruslike transposable elements of Saccharomyces cerevisiae. We showed that regions downstream of the Ty2-917 transcription start site modulate its transcription. One region was located downstream of the transcription initiation site (position 240) and within the first 559 base pairs of the element. This region had a dramatic effect, causing an approximately 1,000-fold increase in steady-state levels of RNA. The region stimulated transcription when placed in either orientation upstream of a heterologous gene, HIS4, lacking its own upstream activation sequence (UAS). We termed this positively acting region an enhancer, by analogy to sites described in higher cells, to distinguish it from yeast UASs which do not function when placed within the transcribed portion of the gene. Though, like some higher eucaryotic enhancers, the Ty2-917 enhancer is located within the transcribed region, it is unlike them in that it occurs within a coding region rather than in an intron. The Ty2-917 enhancer and the Ty2-917 UAS had a synergistic effect on transcription, together stimulating transcription 15-fold over the predicted additive effect. We also identified a site which decreases RNA accumulation, located about 750 base pairs into the element. This site functioned in only one orientation when inserted upstream of the UAS-less heterologous gene. The site was similar to silencers, or negative enhancers, in that it acted to repress transcription from outside the transcribed region, but was distinct in that the function of a canonical silencer was independent of orientation.

scholarly journals Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi

1984 ◽  
Vol 4 (6) ◽  
pp. 1115-1124
Author(s):  
K J Brunke ◽  
J G Anthony ◽  
E J Sternberg ◽  
D P Weeks
Keyword(s):  
Transcription Initiation ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Amino Acid Sequences ◽  
Sequencing Data ◽  
Base Pairs ◽  
Tubulin Gene ◽  
Gene Set ◽  
Tubulin Genes ◽  
Beta 2

The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.

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