Loo, L. W. | Search bioRxiv

bioRxiv2022

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

Hydra actinoporin-like toxin-1 (HALT-1) has been isolated from Hydra magnipapillata and is highly cytolytic against various human cells including erythrocyte. Previously, recombinant HALT-1 (rHALT-1) was expressed in Escherichia coli and purified by the nickel affinity chromatography. In this study, we improved the purification of rHALT-1 by two-step purifications. Bacterial cell lysate containing rHALT-1 was subjected to the sulphopropyl (SP) cation exchange chromatography with different buffers, pHs, and NaCl concentrations. The results indicated that both phosphate and acetate buffers facilitated the strong binding of rHALT-1 to SP resins, and the buffers containing 150 mM and 200 mM NaCl, respectively, removed protein impurities but retain most rHALT-1 in the column. When combining the nickel affinity chromatography and the SP cation exchange chromatography, the purity of rHALT-1 was highly enhanced. In subsequent cytotoxicity assays, 50% of cells could be lysed at [~]18 and [~]22 g/ml of rHALT-1 purified with phosphate and acetate buffers, respectively. O_LIHALT-1 is a soluble -pore-forming toxin of 18.38 kDa. C_LIO_LIrHALT-1 was purified by nickel affinity chromatography followed by SP cation exchange chromatography. C_LIO_LIThe cytotoxicity of purified rHALT-1 using 2-step purifications via either phosphate or acetate buffer was comparable to those previously reported. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/503828v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1b01c1borg.highwire.dtl.DTLVardef@1055147org.highwire.dtl.DTLVardef@1b2eb1org.highwire.dtl.DTLVardef@966fc0_HPS_FORMAT_FIGEXP M_FIG C_FIG Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@82c23forg.highwire.dtl.DTLVardef@9ede5dorg.highwire.dtl.DTLVardef@18e25aorg.highwire.dtl.DTLVardef@1b4b60dorg.highwire.dtl.DTLVardef@11c1eb7_HPS_FORMAT_FIGEXP M_TBL C_TBL

biochemistry↗

bioRxiv2021

Genome-wide Association Study of Pancreatic Fat: The Multiethnic Cohort Adiposity Phenotype Study

Several studies have found associations between higher pancreatic fat content and adverse health outcomes, such as diabetes and the metabolic syndrome, but investigations into the genetic contributions to pancreatic fat are limited. This genome-wide association study, comprised of 804 participants with MRI-assessed pancreatic fat measurements, was conducted in the ethnically diverse Multiethnic Cohort-Adiposity Phenotype Study (MEC-APS). Two genetic variants reaching genome-wide significance, rs73449607 on chromosome 13q21.2 (Beta = -0.67, P = 4.50x10-8) and rs7996760 on chromosome 6q14 (Beta = -0.90, P = 4.91x10-8) were associated with percent pancreatic fat on the log scale. Rs73449607 was most common in the African American population (13%) and rs79967607 was most common in the European American population (6%). Rs73449607 was also suggestively associated with lower risk of type 2 diabetes (OR = 0.95, 95% CI = 0.89-1.00, P = 0.047) in the Population Architecture Genomics and Epidemiology (PAGE) Study and the DIAbetes Genetics Replication and Meta-analysis (DIAGRAM), which included substantial numbers of non-European ancestry participants (53,102 cases and 193,679 controls). Rs73449607 is located in an intergenic region between GSX1 and PLUT, and rs79967607 is in intron 1 of EPM2A. PLUT, a linkRNA, regulates transcription of an adjacent gene, PDX1, that controls beta-cell function in the mature pancreas, and EPM2A encodes the protein laforin, which plays a critical role in regulating glycogen production. If validated, these variants may suggest a genetic component for pancreatic fat and a common etiologic link between pancreatic fat and type 2 diabetes.

cell biology↗