Total PSA Test Performance Evaluation on Cobas Pure and Measurement System Comparison with Different Calibrators.

BACKGROUND: This study investigated the performance evaluation of total prostate specific antigen (tPSA) testing using Cobas Pure integrated solutions system (calibrated against WHO IS 96/670) and the comparison with established measurement systems with different traceability. METHODS: The evaluation was performed in terms of imprecision, linearity, detection limit, and correlation with Alinity i (calibrated against WHO IS 96/670) and Unicel DxI 800 (calibrated against the manufacturer's working calibrators). RESULTS: Within-laboratory reproducibility and repeatability were observed less than 1.2%. Linearity was achieved within the claimed analytical measurement range. The claimed LoB and LoD were experimentally verified. All the correlation coefficients among the assays indicated good correlation, but the significant mean bias with Unicel DxI 800 using a different calibrator were observed. CONCLUSIONS: Since the tPSA calibrators against different traceability is still commercially available, our research could convey the impact of calibration on tPSA results as well as the performance information of a new assay for tPSA.

A New Strategy for Evaluating the Quality of Laboratory Results for Big Data Research: Using External Quality Assessment Survey Data (2010-2020).

Background: To ensure valid results of big data research in the medical field, the input laboratory results need to be of high quality. We aimed to establish a strategy for evaluating the quality of laboratory results suitable for big data research. Methods: We used Korean Association of External Quality Assessment Service (KEQAS) data to retrospectively review multicenter data. Seven measurands were analyzed using commutable materials: HbA1c, creatinine (Cr), total cholesterol (TC), triglyceride (TG), alpha-fetoprotein (AFP), prostate-specific antigen (PSA), and cardiac troponin I (cTnI). These were classified into three groups based on their standardization or harmonization status. HbA1c, Cr, TC, TG, and AFP were analyzed with respect to peer group values. PSA and cTnI were analyzed in separate peer groups according to the calibrator type and manufacturer, respectively. The acceptance rate and absolute percentage bias at the medical decision level were calculated based on biological variation criteria. Results: The acceptance rate (22.5%-100%) varied greatly among the test items, and the mean percentage biases were 0.6%-5.6%, 1.0%-9.6%, and 1.6%-11.3% for all items that satisfied optimum, desirable, and minimum criteria, respectively. Conclusions: The acceptance rate of participants and their external quality assessment (EQA) results exhibited statistically significant differences according to the quality grade for each criterion. Even when they passed the EQA standards, the test results did not guarantee the quality requirements for big data. We suggest that the KEQAS classification can serve as a guide for building big data.

Identification of a Splice Variant (c.5074+3A>C) of BRCA1 by RNA Sequencing and TOPO Cloning.

Grading the pathogenicity of BRCA1/2 variants has great clinical importance in patient treatment as well as in the prevention and screening of hereditary breast and ovarian cancer (HBOC). For accurate evaluation, confirming the splicing effect of a possible splice site variant is crucial. We report a significant splicing variant (c.5074+3A>C) in BRCA1 in a patient with recurrent ovarian cancer. Next-generation sequencing (NGS) of BRCA1/2 from patient's peripheral blood identified the variant, which was strongly suspected of being a splicing mutation based on in silico predictions. Direct RNA analysis yielded multiple transcripts, and TOPO cloning of the complementary DNA (cDNA) and Sanger sequencing revealed an aberrant transcript with an insertion of the first 153 bp of intron 17, and another transcript with the 153 bp insertion along with an exon 18 deletion. A premature termination codon was presumed to be formed by the 153 bp partial intron retention common to the two transcripts. Therefore, BRCA1 c.5074+3A>C was classified as a likely pathogenic variant. Our findings show that active use of functional studies of variants suspected of altered splicing are of great help in classifying them.

Performance evaluation of an amplicon-based next-generation sequencing panel for BRCA1 and BRCA2 variant detection.

BACKGROUND: As next-generation sequencing (NGS) technology matures, various amplicon-based NGS tests for BRCA1/2 genotyping have been introduced. This study was designed to evaluate an NGS test using a newly released amplicon-based panel, AmpliSeq for Illumina BRCA Panel (AmpliSeq panel), for detection of clinically significant BRCA variants, and to compare it to another amplicon-based NGS test confirmed by Sanger sequencing. METHODS: We reviewed BRCA test results done by NGS using the TruSeq Custom Amplicon kit from patients suspected of hereditary breast/ovarian cancer syndrome (HBOC) in 2018. Of those, 96 residual samples with 100 clinically significant variants were included in this study using predefined criteria: 100 variants were distributed throughout the BRCA1 and BRCA2 genes. All target variants were confirmed by Sanger sequencing. Duplicate NGS testing of these samples was performed using the AmpliSeq panel, and the concordance of results from the two amplicon-based NGS tests was assessed. RESULTS: Ninety-nine of 100 variants were detected in duplicate BRCA1/2 genotyping using the AmpliSeq panel (sensitivity, 99%; specificity, 100%). In the discordant case, one variant (BRCA1 c.3627dupA) was found only in repeat 1, but not in repeat 2. Automated nomenclature of all variants, except for two indel variants, was in consensus with Human Genome Variation Society nomenclature. CONCLUSION: Our findings confirm that the analytic performance of the AmpliSeq panel is satisfactory, with high sensitivity and specificity.

GeneReader NGS System Is a Useful Sequencing Platform for Clinical Testing of BRCA1 and BRCA2.

Although next-generation sequencing (NGS) is widely used for BRCA1/2 sequencing analysis, it involves a fragmented workflow along with complex bioinformatic analysis and interpretation. In this study, the performance characteristics and workflow of the GeneReader NGS System (QIAGEN), including BRCA1/2 sequencing, were evaluated. For BRCA1/2 genetic testing, we conducted library preparation, emulsion PCR, and sequencing. QCI Analyze software was used for read alignment, quality control, variant calling, and clinical report generation. GeneReader and Sanger sequencing utilized 63 patients with breast or ovarian cancer for comparison. Reproducibility, precision, variant calling, turnaround time, and hands-on time were evaluated. The read percentage in the on-target regions was 90.5%. More than 99.99% of target regions showed read depths ≥100x. Variants generated from GeneReader showed 100% accuracy compared to the Sanger sequencing results. Annotation with GeneReader showed >99.8% concordance with HGVS nomenclature. Single-nucleotide variations and indel variants showed 100% calling reproducibility; the precision for variant frequency showed a 0.3-3.6% coefficient of variation. Most processes involved hands-off time (3714 min, 88.6% of total run time). The GeneReader NGS System for BRCA1 and BRCA2 testing showed good analytical performance and a short hands-on time. Because of its integrated sample preparation for bioinformatic interpretation, this system is practical for clinical laboratories.

Impact of proactive high-throughput functional assay data on BRCA1 variant interpretation in 3684 patients with breast or ovarian cancer.

The clinical utility of BRCA1/2 genotyping was recently extended from the selection of subjects at high risk for hereditary breast and ovary cancer to the identification of candidates for poly (ADP-ribose) polymerase (PARP) inhibitor treatment. This underscores the importance of accurate interpretation of BRCA1/2 genetic variants and of reducing the number of variants of uncertain significance (VUSs). Two recent studies by Findlay et al. and Starita et al. introduced high-throughput functional assays, and proactively analyzed variants in specific regions regardless of whether they had been previously observed. We retrospectively reviewed all BRCA1 and BRCA2 germline genetic test reports from patients with breast or ovarian cancer examined at Asan Medical Center (Seoul, Korea) between September 2011 and December 2018. Variants were assigned pathogenic or benign strong evidence codes according to the functional classification and were reclassified according to the ACMG/AMP 2015 guidelines. Among 3684 patients with available BRCA1 and BRCA2 germline genetic test reports, 429 unique variants (181 from BRCA1) were identified. Of 34 BRCA1 variants intersecting with the data reported by Findlay et al., three missense single-nucleotide variants from four patients (0.11%, 4/3684) were reclassified from VUSs to likely pathogenic variants. Four variants scored as functional were reclassified into benign or likely benign variants. Three variants that overlapped with the data reported by Starita et al. could not be reclassified. In conclusion, proactive high-throughput functional study data are useful for the reclassification of clinically observed VUSs. Integrating additional evidence, including functional assay results, may help reduce the number of VUSs.

A Simple and Accurate Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Busulfan in Plasma.

OBJECTIVE: Busulfan, frequently used as a conditioning regimen for hematopoietic stem cell transplantation, has a narrow therapeutic range and wide intra-and interpatient variabilities. Therefore, therapeutic drug monitoring of busulfan is necessary to ensure that the drug concentrations of patients are within a targeted therapeutic range. In this study, we developed a simple and accurate method for measuring busulfan concentrations using liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Separation and detection of busulfan was performed using T3 column equipped with LC-MS/MS. Busulfan was isolated from 50 µL human plasma after mixing with busulfan-2H8 (internal standard) solution, calibrator, and quality-control material. The sample was eluted and gradated with a mobile phase composed of ammonium acetate, formic acid, and water or methanol. The busulfan concentration was quantified using a six-point standard curve. Busulfan and busulfan-2H8 were detected in positive-ion multiple-reaction-monitoring mode. According to the Clinical and Laboratory Standards Institute guideline, we verified the precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and carryover. RESULTS: Busulfan and busulfan-2H8 were detected at m/z 264.1>151.1 and 272.2>159.1. The total run time was 3 min. Both intra-and inter-assay coefficients of variation were <3%. The calibration curve was linear at 25-5,000 ng/mL. The LOD and LOQ were 2.5 ng/mL and 25 ng/mL, respectively. The recoveries ranged from 92.0-104.8% and the carryover was-0.02%. CONCLUSIONS: Our method for busulfan reduces total run time and has excellent analytical performance. It will be a useful method for therapeutic drug monitoring of busulfan in clinical laboratories.

Plasma Neutrophil Gelatinase-Associated Lipocalin as a Marker of Tubular Damage in Diabetic Nephropathy.

BACKGROUND: An increase in neutrophil gelatinase-associated lipocalin (NGAL) indicates tubular injury. Diabetic nephropathy causes typical changes in the kidney, characterized by glomerulosclerosis and eventual tubular damage. We validated the usefulness of plasma NGAL (pNGAL) as a biomarker of tubular damage in patients with diabetic nephropathy. METHODS: We included 376 patients with diabetes mellitus (260 patients with chronic renal insufficiency who had not received hemodialysis and 116 hemodialyzed due to diabetic nephropathy) and 24 healthy controls. Patients with chronic renal insufficiency were divided into three groups according to urinary albumin excretion (UAE) levels. pNGAL levels were measured using the Triage NGAL test (Alere, San Diego, CA, USA) and were compared between groups. We also examined whether pNGAL level was related to the degree of albuminuria and cystatin C-based glomerular filtration rate (GFR). RESULTS: Mean pNGAL levels of the healthy controls, chronic renal insufficiency patients with diabetes mellitus, and hemodialyzed patients were 61.9±5.3 ng/mL, 93.4±71.8 ng/mL, and 1,536.9±554.9 ng/mL, respectively. pNGAL level increased significantly in patients with severe albuminuria (P<0.001) and had a moderate correlation with the degree of albuminuria (r=0.467; P<0.001) and GFR (r=0.519; P<0.001). Multivariate regression analysis showed that the pNGAL level was associated with tubular damage independent of patient age, sex, and GFR. CONCLUSIONS: pNGAL level independently reflects the degree of tubular damage in patients with diabetic nephropathy. Measurement of pNGAL, combined with UAE, would enable simultaneous, highly reliable assessments of tubular damage for such patients.

Efficacy and Survival Outcome Associated with the Use of Novel Agents and Autologous Stem Cell Transplantation in Cases of Immunoglobulin D Multiple Myeloma in Korea.

BACKGROUND: Immunoglobulin D multiple myeloma (IgD MM) is characterized by a poor prognosis. Data are lacking on the survival benefits associated with the use of novel agents followed by autologous stem cell transplantation (ASCT) in IgD MM patients. We evaluated the clinical outcomes of induction treatment with novel agents followed by ASCT. METHODS: This was a single-center, retrospective study of 22 IgD MM patients who underwent ASCT between 1995 and 2016. Of these, 10 (45.4%) received novel agents and 12 (54.6%) received nonnovel agents. Clinical features and survival outcomes were examined. RESULTS: Median overall survival (OS) was 37.7 months in the 22 patients. Those in the novel-agents group received bortezomib or thalidomide-based regimens, whereas 91.7% of the nonnovel-agents group received a vincristine-based regimen. The median progression-free survival and OS in the novel-agent/nonnovel-agent groups were 8.3/7.4 and 38.6/12.5 months, respectively. The median OS of patients receiving maintenance therapy was not reached. CONCLUSION: This study showed improved survival outcomes compared to our previous study (37.7 vs. 12 months), suggesting that the use of a novel agent as induction and maintenance therapy may be beneficial in patients with IgD MM who undergo ASCT.

Budget Impact of the Accreditation Program for Clinical Laboratories on Colorectal Cancer Screening via Fecal Immunochemical Testing: Results from the National Cancer Screening Program in Korea.

BACKGROUND: False-positive fecal immunochemical test (FIT) results in the National Cancer Screening Program (NCSP) for colorectal cancer may lead to unnecessary procedures, such as colonoscopies, increasing the medical costs. We estimated reductions in the cost of National Health Insurance according to the accreditation status of screening facilities participating in the NCSP for colorectal cancer. METHODS: We used data collected between 2007 and 2010 from NCSP and the Korea Central Cancer Registry to identify patients with colorectal cancer. We also ascertained the history of the accreditation of each facility by the Korean Laboratory Accreditation Program (KLAP). Budget impact was defined as a reduction in medical costs achieved when the false-positive rate of the non-accredited facilities decreased relative to that of the accredited facilities. RESULTS: A total of 3,285 screening facilities participated in the NCSP. Of these, 196 were accredited by the KLAP. The false-positive rate of the accredited facilities was 2.47%, and that of the non-accredited facilities was 6.83%. Medical costs were estimated to be reduced by approximately 19 million US dollars (USD), and the cost of detecting one case of colorectal cancer was estimated to decrease from 9,212 USD to 7,332 USD if the false-positive rate of non-accredited facilities were decreased to that of the accredited facilities. Clinics were estimated to have the largest associated cost reduction. CONCLUSIONS: Quality assurance in clinical laboratories could lower false-positive rates and prevent the use of unnecessary procedures, ensuring patient safety and increasing the cost-effectiveness of FIT screening in the NCSP for colorectal cancer.

Evaluation of the 1B Equation to Estimate Glomerular Filtration Rate in Pediatric Patients with Cancer.

The 1B equation is recommended for calculating the glomerular filtration rate (GFR) in children. Since few reports have evaluated the performance of the 1B equation, we investigated the performance of estimated GFR (eGFR) equations with the blood urea nitrogen (BUN) variable for pediatric cancer patients. In total, 203 children with cancer who underwent measured GFR (mGFR) assessment were enrolled. The median (range) mGFR and eGFR calculated using the updated Schwartz equation were 118 (43-241) and 135 (34-257) mL/min/1.73 m², respectively. The bias, precision (root mean square error [RMSE]), and accuracy (P30, mGFR±30%) of three eGFR equations including updated Schwartz, 1B, and full age spectrum (FAS) were compared. The median bias (mL/min/1.73 m²) was: updated Schwartz, 8.5; 1B, -9.0; and FAS, 4.2. The biases for all three eGFR equations were significantly different from zero. The P30 was: updated Schwartz, 63.5%; 1B, 66.0%; and FAS, 66.0%. The RMSE was the lowest for the 1B equation (40.4), followed by FAS (42.3), and updated Schwartz (45.5). The median eGFR/mGFR ratio for the eGFR equations decreased with age and reduced kidney functions (i.e., increased creatinine and BUN concentrations). The bias may be further reduced by using the average from two equations, such as the updated Schwartz and 1B, or FAS equation, rather than using the updated Schwartz or 1B equation alone. The use of the 1B equation may underestimate the GFR. Using creatinine and BUN variables in the eGFR equation may yield a more accurate estimate of the GFR in pediatric cancer patients.

Efficient reporting of the estimated glomerular filtration rate without height in pediatric patients with cancer.

BACKGROUND: The updated bedside Schwartz equation requires constant, serum creatinine concentration and height measurements to calculate the estimated glomerular filtration rate (eGFR) in pediatric patients. Unlike the serum creatinine levels, obtaining height information from the laboratory information system (LIS) is not always possible in a clinical laboratory. Recently, the height-independent eGFR equation, the full age spectrum (FAS) equation, has been introduced. We evaluated the performance of height-independent eGFR equation in Korean children with cancer. METHODS: A total of 250 children who underwent chromium-51-ethylenediamine tetra acetic-acid (51Cr-EDTA)-based glomerular filtration rate (GFR) measurements were enrolled. The 51Cr-EDTA GFR was used as the reference GFR. The bias (eGFR - measured GFR), precision (root mean square error [RMSE]) and accuracy (P30) of the FAS equations were compared to those of the updated Schwartz equation. P30 was defined as the percentage of patients whose eGFR was within ±30% of the measured GFR. RESULTS: The FAS equation showed significantly lower bias (mL/min/1.73 m2) than the updated Schwartz equation (4.2 vs. 8.7, p<0.001). The RMSE and P30 were: updated Schwartz of 43.8 and 64.4%, respectively, and FAS of 42.7 and 66.8%, respectively. CONCLUSIONS: The height-independent eGFR-FAS equation was less biased and as accurate as the updated Schwartz equation in Korean children. The use of the height-independent eGFR equation will allow for efficient reporting of eGFR through the LIS in clinical laboratories.

A new strategy for calculating the risk of ovarian malignancy algorithm (ROMA).

BACKGROUND: Reliable quantitative measurements of HE4 and CA125 levels are required to calculate the risk of ovarian malignancy algorithm (ROMA) value. We suggest a new reporting strategy for interpreting ROMA values based on analytical measurement range (AMR) and qualified-intervals of the HE4 and CA125 results. METHODS: HE4 and CA125 assays from Abbott and Roche were used. The AMRs and the qualified-intervals were as follows: Architect HE4 assay, 20-1500 and 17.2-2637.8 pmol/L; Architect CA125 II assay, 1-1000 and 3.9-14,163.0 U/mL; Elecsys HE4 assay, 15-1500 and 28.8-3847 pmol/L; Elecsys CA125 II assay, 0.6-5000 and 6.5-5000 U/mL. These values were used to simulate the ROMA values. RESULTS: Reporting algorithm for the ROMA value could be classified into three categories. (1) If quantitative HE4 and CA125 levels are reliable, the numerical ROMA value can be reported. (2) If HE4 value is <20 and <28.8 for Abbott and Roche in premenopausal woman, the ROMA value should be reported as "low risk" regardless of the CA125 result. In postmenopausal woman, however, it should be reported as "low risk" (CA125<203.0 and <165.8 for Abbott and Roche) or "undetermined" (vice-versa value). (3) If CA125 value is <3.9 and <6.5 for Abbott and Roche, it should be reported as "low risk" (premenopausal HE4<51.5 and <62.2, postmenopausal HE4<323.0 and <281.5 for Abbott and Roche) or "undetermined" (vice-versa value). CONCLUSIONS: New reporting strategy will provide more informative reporting of ROMA values in clinical practice.

Comparison of Thyroglobulin Measurements Using Three Different Immunoassay Kits: A BRAMHS Tg-Plus RIA Kit, a BRAMHS hTg Sensitive Kryptor Kit, and a Beckman Coulter ACCESS Immunoassay Kit.

BACKGROUND: Second-generation thyroglobulin immunometric assays (Tg-IMAs) have been developed with improved sensitivity. Our aim was to compare the diagnostic value of Tg-IMA measurements using a Kryptor (BRAHMS AG) kit (Tg-K) and an ACCESS (Beckman Coulter) kit (Tg-A) with that of the first-generation Tg measurement using a Tg-plus (BRAHMS AG) kit (Tg+). METHODS: We enrolled 82 differentiated thyroid cancer patients who underwent total thyroidectomy with radioactive iodine remnant ablation and who underwent diagnostic whole body scan using recombinant human thyroid stimulating hormone (rhTSH). The Tg+, Tg-K, and Tg-A were measured before rhTSH administration during levothyroxine treatment (suppressed Tg) from the same sample. Serum Tg+ was measured after rhTSH stimulation (stimulated Tg). RESULTS: Suppressed Tg+ was more significantly correlated with suppressed Tg-K (R²=0.919, P<0.001) than with suppressed Tg-A (R²=0.536, P<0.001). The optimal cut-off values of suppressed Tg+, Tg-K, and Tg-A for predicting stimulated Tg+ of 1 ng/mL were 0.3, 0.2, and 0.2 ng/mL, respectively. The sensitivity, specificity, and accuracy of suppressed Tg+ were 67%, 100%, and 90%, respectively; those of suppressed Tg-K were 83%, 90%, and 88%; those of suppressed Tg-A were 96%, 82%, and 87%, respectively. The positive predictive and negative predictive values of Tg+ were 100% and 87%, respectively; those of Tg-K were 79% and 92%; and those of Tg-A were 73% and 98%. CONCLUSION: We could not clearly demonstrate which kit had better diagnostic performance after comparison of first-generation Tg measurements with Tg-IMA measurements. Also, there were kit-to-kit variations between Tg-IMA kits. Suppressed Tg measured by Tg-IMA was insufficient to completely substitute for a stimulated Tg measurement.

Development and validation of the Korean version of CKD-EPI equation to estimate glomerular filtration rate.

BACKGROUND: The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was derived mostly from Caucasian, African-American, and Hispanic populations, whereas Asian populations were excluded. The aim of this study was to develop and validate a Korean version of the CKD-EPI equation. METHODS: The study enrolled 960 individuals 18years old and older who underwent chromium-51-ethylenediaminetetraacetic-acid-based glomerular filtration rate (GFR) measurements. They were divided randomly into two groups: a development set (n=768, 80%) and a validation set (n=192, 20%). The Korean CKD-EPI equation was developed using a non-linear mixed-effect model. The performance of the equation was evaluated by calculating the bias (estimated GFR-measured GFR). The ±10% (P10) and ±30% (P30) accuracies and root mean square errors (RMSEs) of the original and Korean CKD-EPI equations were compared. RESULTS: The Korean CKD-EPI equation was as follows: male, serum creatinine (Scr) ≤80µmol/L, GFR=141×(Scr/0.9)(-0.642)×(0.993)(Age); male, Scr>80µmol/L, GFR=141×(Scr/0.9)(-1.128)×(0.993)(Age); female, Scr≤62µmol/L, GFR=144×(Scr/0.7)(-0.465)×(0.993)(Age); female, Scr>62µmol/L, GFR=144×(Scr/0.7)(-1.382)×(0.993)(Age). The mean bias (mL/min/1.73m(2)) of the original CKD-EPI equation was -3.0±13.5 and that of the Korean CKD-EPI equation -2.3±13.3. The P10 and P30 of the original CKD-EPI equation were 33.9% and 82.8%; for the Korean CKD-EPI equation, the corresponding values were 35.4% and 85.9%. The RMSEs of the original and Korean CKD-EPI equations were 13.7 and 13.5, respectively. CONCLUSIONS: The overall analytical performance of the Korean CKD-EPI equation was equivalent to that of the original CKD-EPI equation. The original CKD-EPI equation is therefore also valid for the Korean population.

Method evaluation of pepsinogen I/II assay based on chemiluminescent immunoassays and comparison with other test methods.

BACKGROUND: Serum pepsinogen (PG) I and the PG I/PG II ratio have been used for atrophic gastritis (AG) diagnosis for decades. Low levels of PG I and/or PG I/PG II are closely related to AG and predict the risk of gastric cancer. We evaluated the performance of the chemiluminescent immunoassay-based Architect Pepsinogen I/II assay. METHODS: The evaluation consisted of determination of the precision, linearity, limit of blank (LoB), limit of detection (LoD) and method comparison with Eiken and Biohit assays. RESULTS: The total CVs were below 5% for both PG I and PG II. Acceptable linearity was observed for PG I and PG II in their respective reportable ranges. The PG I LoB was 0.317ng/mL and the PG II LoB was 0.418ng/mL, and LoDs were 0.412ng/mL and 0.497ng/mL, respectively. Correlation analysis indicated that results of the Architect assay were comparable to those of the Eiken and Biohit assays, but the three methods lead to different estimations of the cancer risk. CONCLUSION: The overall analytical performance of Architect Pepsinogen I/II assay is acceptable for the detection of patients with suspected AG. The categorization results of gastric cancer risk showed some difference among test methods suggesting the need for harmonization among the methods from vendors.

Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II.

Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.