Integrated Strategies for Enhancing the Expression of Chitosanase AqCoA in the Pichia Pastoris by A Novel High-Copy Plasmid PMC-GAP

2021 ◽  
Author(s):  
Yanxin Wang ◽  
Yuqiang Zhao ◽  
Xue Luo ◽  
Zhoukun Li ◽  
Xianfeng Ye ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Protein Expression ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Maximum Activity ◽  
Gene Copy ◽  
Foreign Protein ◽  
Expression Levels ◽  
Model Combining ◽  
Enhanced Expression

Abstract In our previous study, the chitosanase AqCoA and its products chitooligosaccharides exhibited significant application in fungal disease protection. In this study, to enhance the expression of AqCoA, we obtained various strains with multi-copy by a novel plasmid pMC-GAP with stable transformation ability in Pichia pastoris and built an integrated model combining the gene copy number, the chaperones and protein production of AqCoA. In terms of gene dosage, the highest enzyme activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that in strain with only one copy (0.18 U/ml). In addition, we found the chaperone like PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1 and YDJ1/SSO2 pairs synergistically increased by 61%, 31% and 42% in expression levels of the strain GAP-1AqCoA. Finally, we investigated the effect co-expression of gene copy and chaperones on protein expression. The maximum activity reached 2.319 U/ml by the strain with six chaperones intergrant plus sixteen copies, which was 13-fold higher than that by the control strain with only one copy (GAP-1AqCoA). Co-expression of gene dosage and chaperones significantly enhanced expression levels of AqCoA, which presented a powerful tool to improve foreign protein expression.

scholarly journals Insulin-like Growth Factor Receptor 1 (IGF1R) Gene Copy Number Is Associated With Survival in Operable Non–Small-Cell Lung Cancer: A Comparison Between IGF1R Fluorescent In Situ Hybridization, Protein Expression, and mRNA Expression

2010 ◽  
Vol 28 (13) ◽  
pp. 2174-2180 ◽  
Author(s):  
Rafal Dziadziuszko ◽  
Daniel T. Merrick ◽  
Samir E. Witta ◽  
Adelita D. Mendoza ◽  
Barbara Szostakiewicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Protein Expression ◽  
Squamous Cell ◽  
Copy Number ◽  
Gene Copy Number ◽  
Growth Factor Receptor ◽  
Gene Copy ◽  
Cell Versus

PurposeThe purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non–small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis.Patients and MethodsOne hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181).ResultsIGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis.ConclusionIGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.

Gene Expression Profiling of Hyperdiploid Multiple Reveal Complex Gene Dosage Effects and an mRNA Translation/Protein Synthesis Signature.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1538-1538
Author(s):  
Wee-Joo Chng ◽  
Scott Van Wier ◽  
Gregory Ahmann ◽  
Tammy Price-Troska ◽  
Kim Henderson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Differentially Expressed Genes ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Mrna Translation ◽  
Differentially Expressed ◽  
Gene Copy ◽  
Gene Dosage Effect ◽  
Expression Of Genes

Abstract Hyperdiploid MM (H-MM), characterized by recurrent trisomies constitute about 50% of MM, yet very little is known about its pathogenesis and oncogenic mechanisms. Studies in leukemia and solid tumors have shown gene dosage effect of aneuploidy on gene expression. To determine the possible gene dosage effect and deregulated cellular program in H-MM we undertook a gene expression study of CD138-enriched plasma-cell RNA from 53 hyperdiploid and 37 non-hyperdiploid MM (NH-MM) patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Gene expression data was analyzed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Genes differentially expressed between H-MM and NH-MM were obtained by t-test (p&lt;0.01). The majority of the differentially expressed genes (57%) were under-expressed in H-MM. Genes located on the commonly trisomic chromosomes were mostly (but not always) over-expressed in H-MM and constitute 76% of over-expressed genes. Chromosome 1 contained the most differentially expressed genes (17%) followed by chromosome 12 (9%), and 19 (8%). To examine the relationship of gene copy number to gene expression, we examined the expression of genes on chromosomes 9 and 15 in subjects with 2 copies (15 normal control and 20 NH-MM) and 3 copies (12 H-MM) of each chromosome as detected by interphase FISH. We then derived a ratio of the mean expression of each gene on these chromosomes between patients with 3 copies and 2 copies of the chromosome. If a simple relationship exists between gene expression and gene copy number, one would expect the ratio of expression of most genes on these two chromosomes to be about 1.5 in H-MM compared to NH-MM. However, many genes have ratios either higher than 2 or lower than 0.5. Furthermore, when the heterogeneity of cells with underlying trisomies is taken into consideration by correcting the ratio for the number of cells with trisomies, the actual ratio is always lower than the expected ratio. When the expression of genes on a chromosome was compressed to a median value, this value was always higher in the trisomic chromosomes for H-MM compared to NH-MM. The data suggests that although gene dosage influence gene expression, the relationship is complex and some genes are more gene dosage dependent than others. Amongst the differentially expressed genes with known function, 33% are involved in mRNA translation/protein synthesis. Of note, 37 of the top 100 differentially expressed genes are involved in these processes. In particular, 60 ribosomal protein (RP) genes are significantly (p&lt;0.05) upregulated in H-MM. This signature in H-MM is not associated with increase proliferation as measured by PCLI. This predominant signature suggests that deregulated protein synthesis may be important for the biology of H-MM. Many of these RPs are involved in the synthesis of product of oncogenic pathways (e.g. MYC, NF-KB pathways) and may mediate the growth and survival of tumor cells. It is therefore possible that these tumor cells may be sensitive to the disruption of mRNA translation/protein synthesis. Targeting the mTOR pathway with rapamycin may therefore be useful for treatment of H-MM.

Gene copy number of epidermal growth factor receptor (EGFR) by fluorescent in situ hybridization (FISH) in head and neck squamous cell carcinomas (HNSCC) is closely correlated with EGFR protein levels assessed by automated quantitative protein analysis (AQUA)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 6023-6023
Author(s):  
P. Weinberger ◽  
A. Psyrri ◽  
P. Kountourakis ◽  
T. Rampias ◽  
C. Sasaki ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Protein Expression ◽  
Protein Content ◽  
Gene Amplification ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Egfr Gene ◽  
Protein Levels

6023 Background: EGFR overexpression correlates with recurrence and with treatment resistance in HNSCC. The mechanisms of EGFR protein overexpression are poorly understood. Nonetheless, previous investigators have not demonstrated a correlation between EGFR gene copy number and protein content, using conventional immunohistochemistry (IHC). The aim of this study was to evaluate the relationship of EGFR gene copy number and protein expression utilizing fluorescence in situ hybridization (FISH) and AQUA, a novel, immunohistochemical method of automated quantitative in situ proteomic analysis which permits subcellular localization. Methods: A tissue microarray composed of 137 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis/Abbot) and EGFR protein expression (DAKO antibody) using AQUA analysis of EGFR staining scored on a scale of 0–255 and by conventional IHC. Agreement was assessed using kappa. Results: Sixteen (15%) of one-hundred six tumors with FISH results demonstrated EGFR high polysomy and/or gene amplification (FISH+). AQUA demonstrated a range of 3.6–102.2; protein levels assessed by AQUA in the FISH amplified cases were significantly higher (p =0.008) than in the FISH non- amplified ones. Using the EGFR 75th percentile as a cut-off, AQUA and FISH showed significant agreement (percentage of overall agreement 82%, kappa=0.458, p=0.003). To the contrary there was no concordance between FISH and conventional IHC results in this series. Conclusions: The discrepancy between EGFR gene amplification rate and protein expression by IHC reported previously may be due to the limitations and nonquantitative nature of conventional IHC. EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. No significant financial relationships to disclose.

Protein expression (PE) and increased IGF1R gene copy number (GCN) in small cell lung cancer (SCLC).

2010 ◽  
Vol 28 (15_suppl) ◽  
pp. 10584-10584
Author(s):  
A. Badzio ◽  
M. W. Wynes ◽  
R. Dziadziuszko ◽  
D. Merrick ◽  
M. Pardo ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Protein Expression ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Copy Number ◽  
Gene Copy Number ◽  
Small Cell ◽  
Gene Copy ◽  
Small Cell Lung

Androgen Receptor Gene Copy Number and Protein Expression in Treatment-Naïve Prostate Cancer

2017 ◽  
Vol 99 (2) ◽  
pp. 222-228 ◽  
Author(s):  
Filip Poelaert ◽  
Candy Kumps ◽  
Nicolaas Lumen ◽  
Stephanie Verschuere ◽  
Louis Libbrecht ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Protein Expression ◽  
Copy Number ◽  
Receptor Gene ◽  
Gene Copy Number ◽  
Androgen Receptor Gene ◽  
Gene Copy ◽  
Treatment Naïve

scholarly journals Rapid Real-Time Fluorescent PCR Gene Dosage Test for the Diagnosis of DNA Duplications and Deletions

2000 ◽  
Vol 46 (10) ◽  
pp. 1574-1582 ◽  
Author(s):  
Clara Ruiz-Ponte ◽  
Lourdes Loidi ◽  
Ana Vega ◽  
Angel Carracedo ◽  
Francisco Barros
Keyword(s):  
Real Time ◽  
Capillary Tube ◽  
Gene Dosage ◽  
Melting Curve ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Target Sequence ◽  
Hereditary Neuropathy ◽  
Target Dna ◽  
Fluorescent Pcr

Abstract Background: Current methods to determine gene dosage are time-consuming and labor-intensive. We describe a new and rapid method to assess gene copy number for identification of DNA duplications or deletions occurring in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively. Methods: We studied 16 patients with HNPP, 4 with CMT1A, and 49 control subjects. We used real-time PCR on the LightCycler system with use of a single capillary tube and no post-PCR handling. A polymorphic fragment of the PMP22 gene was amplified to determine gene dosage for heterozygous samples. The presence of two alleles was used to indicate that no deletion was present in HNPP samples. The ratio obtained between the areas under each allele melting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitive gene dosage test, where the ratio between the areas under the melting curves of the target DNA of samples and of the competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. Samples from HNPP, CMT1A, and controls were analyzed. Results: Area ratios were ∼0.6, 1.0, and 2.0 for HNPP, control, and CMT1A samples, respectively. The results agreed with those obtained by Southern blotting and microsatellite analysis in the same samples. Conclusions: Direct and competitive real-time fluorescent PCR can differentiate one, two, or three copies of the target DNA. The method described is sensitive and accurate for detection of CMT1A duplications and HNPP deletions and is faster and easier than current methods.

scholarly journals Genome instability drives epistatic adaptation in the human pathogen Leishmania

2021 ◽  
Vol 118 (51) ◽  
pp. e2113744118
Author(s):  
Giovanni Bussotti ◽  
Laura Piel ◽  
Pascale Pescher ◽  
Malgorzata A. Domagalska ◽  
K. Shanmugha Rajan ◽  
...  
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Biomarker Discovery ◽  
Genome Instability ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Snorna Gene ◽  
Ideal System

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2′-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

Sign in / Sign up

Export Citation Format

Share Document