Of MBP fusion proteins in E. coli. Conversely, because solubility enhancement

Of MBP fusion order GDC-0032 proteins in E. coli. Conversely, because solubility enhancement is an intrinsic property of MBP, the production of MBP fusion proteins in eukaryotic expression systems might yield favorable results. Recently, MBP has also been used to maintain proteins that contain disulfide-bonds in a soluble state in the E. coli cytoplasm so that they could be acted upon by appropriate redox enzymes that were co-expressed in the same cellular compartment [47]. It seems likely that additional ways of exploiting the “holdase” activity of MBP for the production of recombinant proteins will be forthcoming.Figure S2 Interaction of NusA fusion proteins with GroEL/S. (A) Lysed cells co-expressing His6-NusA-GFP and either wild-type GroE or the GroE3? variant are shown under blue or white light illumination. Cells co-expressing GroE3? fluoresce more intensely than cells co-expressing wild-type GroE as a result of enhanced GFP folding. Cells expressing only the His6-NusA-GFP fusion protein are shown on the left. (B) SDSPAGE analysis of total and soluble proteins from the cells in (A). T, total intracellular protein; S, soluble intracellular protein. (TIF) Figure S3 Enzymatic activity from cells co-expressing GroEL/S and His6-MBP-fusions. (A) G3PDH activity. (B) DHFR activity. The data with error bars are expressed as mean 6 standard error of the mean (n = 3). Extracts from “wild-type” E. coli K-12 were prepared by sonication from equal amounts of cells expressing GroEL and GroES (pGroEL/S) or His6-MBP-fusions (G3PDH or DHFR) alone, or fusion proteins with GroEL/S (pGroEL/S+His6-MBP-G3PDH or His6-MBP-DHFR). The extracts 1480666 were centrifuged at 14000 g for 10 min, and the soluble fraction was assayed for enzymatic activity. (TIF)Supporting InformationFigure S1 Copurification of GroEL with natively purified MBP fusions on an affinity (IMAC) column. (A) Western blot using anti-GroEL antibody. Lane 1, His6-MBPG3PDH; lane 2, His6-MBP-DHFR; lane 3, His6-MBP; lane 4, 1676428 purified GroEL. (B) SDS-PAGE analysis of the above samples (loading same as above). (TIF)The Mechanism of Solubility Enhancement by MBPAcknowledgmentsWe thank the staff of the Biophysics Resource in the Structural Biophysics Laboratory, Frederick National Laboratory, for assistance with spectrofluorometry measurements. We are also grateful to the FNL Scientific Publications, Graphics and Media service for their help with the preparation of Figure 7. The content of this publication does not necessarily reflect the views or policies of the Department of Health andHuman Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the US Government.Author ContributionsConceived and designed the experiments: SRK DSW. Performed the experiments: SRK. Analyzed the data: SRK DSW. Wrote the paper: SRK DSW.
A neutralizing antibody (NAb) response of sufficient duration and magnitude is considered an important part of a successful HIV vaccine [1?]. Numerous studies have demonstrated sterilizing protection by NAbs against challenge with HMPL-013 site simianhuman immunodeficiency virus (SHIV) in nonhuman primate models [4?], and the selection pressure that NAbs exert on the virus during natural infection in humans [8?1]. These observations overwhelmingly suggest that the presence of similar types of NAbs elicited by a vaccine would be beneficial to the vaccinee. The only target for neutralizing antibodies on HIV is the virally encoded envelope glycoprotein (Env) spike. The funct.Of MBP fusion proteins in E. coli. Conversely, because solubility enhancement is an intrinsic property of MBP, the production of MBP fusion proteins in eukaryotic expression systems might yield favorable results. Recently, MBP has also been used to maintain proteins that contain disulfide-bonds in a soluble state in the E. coli cytoplasm so that they could be acted upon by appropriate redox enzymes that were co-expressed in the same cellular compartment [47]. It seems likely that additional ways of exploiting the “holdase” activity of MBP for the production of recombinant proteins will be forthcoming.Figure S2 Interaction of NusA fusion proteins with GroEL/S. (A) Lysed cells co-expressing His6-NusA-GFP and either wild-type GroE or the GroE3? variant are shown under blue or white light illumination. Cells co-expressing GroE3? fluoresce more intensely than cells co-expressing wild-type GroE as a result of enhanced GFP folding. Cells expressing only the His6-NusA-GFP fusion protein are shown on the left. (B) SDSPAGE analysis of total and soluble proteins from the cells in (A). T, total intracellular protein; S, soluble intracellular protein. (TIF) Figure S3 Enzymatic activity from cells co-expressing GroEL/S and His6-MBP-fusions. (A) G3PDH activity. (B) DHFR activity. The data with error bars are expressed as mean 6 standard error of the mean (n = 3). Extracts from “wild-type” E. coli K-12 were prepared by sonication from equal amounts of cells expressing GroEL and GroES (pGroEL/S) or His6-MBP-fusions (G3PDH or DHFR) alone, or fusion proteins with GroEL/S (pGroEL/S+His6-MBP-G3PDH or His6-MBP-DHFR). The extracts 1480666 were centrifuged at 14000 g for 10 min, and the soluble fraction was assayed for enzymatic activity. (TIF)Supporting InformationFigure S1 Copurification of GroEL with natively purified MBP fusions on an affinity (IMAC) column. (A) Western blot using anti-GroEL antibody. Lane 1, His6-MBPG3PDH; lane 2, His6-MBP-DHFR; lane 3, His6-MBP; lane 4, 1676428 purified GroEL. (B) SDS-PAGE analysis of the above samples (loading same as above). (TIF)The Mechanism of Solubility Enhancement by MBPAcknowledgmentsWe thank the staff of the Biophysics Resource in the Structural Biophysics Laboratory, Frederick National Laboratory, for assistance with spectrofluorometry measurements. We are also grateful to the FNL Scientific Publications, Graphics and Media service for their help with the preparation of Figure 7. The content of this publication does not necessarily reflect the views or policies of the Department of Health andHuman Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the US Government.Author ContributionsConceived and designed the experiments: SRK DSW. Performed the experiments: SRK. Analyzed the data: SRK DSW. Wrote the paper: SRK DSW.
A neutralizing antibody (NAb) response of sufficient duration and magnitude is considered an important part of a successful HIV vaccine [1?]. Numerous studies have demonstrated sterilizing protection by NAbs against challenge with simianhuman immunodeficiency virus (SHIV) in nonhuman primate models [4?], and the selection pressure that NAbs exert on the virus during natural infection in humans [8?1]. These observations overwhelmingly suggest that the presence of similar types of NAbs elicited by a vaccine would be beneficial to the vaccinee. The only target for neutralizing antibodies on HIV is the virally encoded envelope glycoprotein (Env) spike. The funct.