(2) Selected Abstracts & Publications from Some Sub-Fields of Our Work.

A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter. Nallaseth FS, Anderson S. Microbial Cell Factories       NCBINCBI Logo     Skip to main content     Skip to navigation     Resources     How To     About NCBI Accesskeys  Sign in to NCBI PubMed US National Library of Medicine National Institutes of Health Search database Search term Clear input      Advanced     Help  Result Filters Display Settings:      Abstract  Send to: Microb Cell Fact. 2013 Apr 19;12:36. doi: 10.1186/1475-2859-12-36. - Abstract BACKGROUND:  To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. RESULTS:  Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. CONCLUSIONS:  The modified immuno-chromogenic screen is sensitive, specific and has led to the isolation of mutants E(M) over-secreting the α-sec-EAP reporter protein by a minimum of 50 fold higher levels than that exported by non-mutagenized E(P) parental strains. Unselected proteins were also over-secreted.

Crystallographic analysis of human cathepsin K-C4S complexes. - Structure-activity analysis of cathepsin K/chondroitin 4-sulfate interactions. Cherny MM, Lecaille F, Kienitz Deceased M, Nallaseth FS, Li Z, James MN, Bromme D.

http://www.ncbi.nlm.nih.gov/pubmed/14698633?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum - Brömme D, Nallaseth FS, Turk B. Production and activation of recombinant papain-like cysteine proteases.  

http://www.ncbi.nlm.nih.gov/pubmed/15899853?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum - Mullen JR, Nallaseth FS, Lan YQ, Slagle CE, Brill SJ. Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex. 

http://www.ncbi.nlm.nih.gov/pubmed/1605860?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum - Hornsby PJ, Yang L, Raju SG, Maghsoudlou SS, Lala DS, Nallaseth FS. Demethylation of specific sites in the 5'-flanking region of the CYP17 genes when bovine adrenocortical cells are placed in culture. 

http://www.ncbi.nlm.nih.gov/pubmed/1721870?ordinalpos=8&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum - Burhans WC, Vassilev LT, Wu J, Sogo JM, Nallaseth FS, DePamphilis ML. Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation. 

http://www.ncbi.nlm.nih.gov/pubmed/3737402?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum - Nallaseth FS, Dewey MJ. Moderately repeated mouse Y chromosomal sequence families present distinct types of organization and evolutionary change. 

http://www.ncbi.nlm.nih.gov/pubmed/7956062?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum - Gilbert DM, Miyazawa H, Nallaseth FS, Ortega JM, Blow JJ, DePamphilis ML. Site-specific initiation of DNA replication in metazoan chromosomes and the role of nuclear organization.

http://www.ncbi.nlm.nih.gov/pubmed/8151772?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum - Nallaseth FS, DePamphilis ML. Papillomavirus contains cis-acting sequences that can suppress but not regulate origins of DNA replication.