| Title: | 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 |
| Author(s): | Nallaseth FS; Anderson S; |
| Address: | "Department for Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. ferez.nallaseth@gmail.com" |
| ISSN/ISBN: | 1475-2859 (Electronic) 1475-2859 (Linking) |
| 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 alpha factor pheromone through cellular PHO1/KEX2 secretory processing signals as the alpha-sec-EAP reporter protein. Direct chromogenic staining for alpha-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 alpha-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 alpha-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 alpha-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 alpha-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 alpha-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" |
| Keywords: | Aldehyde Oxidase/genetics Alkaline Phosphatase/chemistry/genetics/*metabolism Escherichia coli/*enzymology Escherichia coli Proteins/chemistry/genetics/*metabolism Genetic Loci Genotype Mutagenesis Phenotype Pichia/genetics/*metabolism Recombinant Protein; |
| Notes: | "MedlineNallaseth, Ferez S Anderson, Stephen eng England 2013/04/23 Microb Cell Fact. 2013 Apr 19; 12:36. doi: 10.1186/1475-2859-12-36" |
