Crystal structure of native Anopheles gambiae serpin-2, a negative regulator of melanization in mosquitoes
METHODS
Protein production and purification
The pET28a_His-SRPN2 plasmid was used to transform Escherichia coli BL-21 DE3 pRare; two colonies were picked and inoculated in 15 mL of LB medium containing 50 μg mL−1 kanamycin and 25 mg mL−1 chloramphenicol. A starter culture was transferred to 1.5 L of LB medium containing antibiotics and grown to an OD600 ∼0.8 at 37°C. Induction was carried out with 0.1 mM IPTG at 15°C overnight with shaking. Cells were harvested via centrifugation at 8500 rpm for 10 min. Cell pellets were resuspended in 50 mL of Buffer A (50 mM NaCl, 20 mM Tris-HCl, pH of 8.0) supplemented with protease inhibitor cocktail (Roche) and lysed by sonication. The insoluble material was pelleted by centrifugation at 19,000 rpm for 30 min and the clarified lysate was loaded onto a 5 mL HisTrap HP column. All purification steps were conducted using an AKTA Xpress purification system (GE Healthcare) at 4°C. Non-specifically bound proteins were washed using 10% Buffer B (500 mM Imidazole, 50 mM NaCl, 50 mM Tris-HCl, pH 8.0). Elution was carried out with a linear gradient from 10 to 100% Buffer B and all elution peaks of interest were collected and analyzed by SDS-PAGE. Fractions containing SRPN2 were pooled and loaded onto a HiTrap Q HP anion exchange column equilibrated with Buffer A. Elution was carried out with a linear gradient from 0 to 100% Buffer C (1M NaCl, 20 mM Tris, pH 8.0) and the purity was analyzed by SDS-PAGE. SRPN2 fractions, which eluted at ∼50% Buffer C, were pooled and concentrated to 6.2 mg mL−1 for crystallization screening.
Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18
Sci Rep. 2015 Jul 7;5:11863. doi: 10.1038/srep11863.
Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18
Sci Rep. 2015 Jul 7;5:11863. doi: 10.1038/srep11863.
Construction, expression, and purification of serpin18
The open reading frame of SERPIN18 without the signal peptide was amplified by PCR, using B. mori(strain p50, Dazao) silk gland cDNA as the template, and cloned into a pET28a-derived vector. This construct with an N-terminal hexahistidine-tag was transformed into E. coli BL21 (DE3) (Novagen, Madison, WI) and induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) at 16 °C for 20 h when OD600nm reached 0.6. Cells were harvested by centrifugation at 8000 g for 10 min and resuspended in lysis buffer (20 mM Tris-HCl, pH 6.8, 200 mM NaCl). After 5 min of sonication and centrifugation at 12,000 g for 25 min, the supernatant containing the soluble target protein was loaded onto a HiTrap nickel-chelating column (GE Healthcare) that had been equilibrated with binding buffer (20 mM Tris-HCl, pH 6.8, 200 mM NaCl). The target protein was eluted with 250 mM imidazole buffer and further loaded onto a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 6.8, 50 mM NaCl. Fractions containing the target protein were pooled and concentrated to 20 mg/mL. The purity of protein was estimated on SDS-PAGE and the protein sample was stored at −80 °C. The mutant proteins were expressed, purified, and stored using the same protocol as the wild-type protein.
Selenomethionine (SeMet)-labeled serpin18 proteins were expressed in E. coli strain B834 (DE3) (Novagen). A culture of transformed cells was inoculated into LB medium and incubated at 37 °C overnight. The cells were cultured in SeMet medium (M9 medium with 25 mg/L SeMet and other essential amino acids at 50 mg/L) to an OD600nm of 0.6–0.8. Protein expression and purification were the same as those for the native protein.
Towards Engineering Hormone-Binding Globulins as Drug Delivery Agents
Towards Engineering Hormone-Binding Globulins as Drug Delivery Agents
PLoS One. 2014; 9(11): e113402.
Published online 2014 Nov 26. doi: 10.1371/journal.pone.0113402
Materials and Methods
Recombinant CBG
Wild type and engineered human CBG were expressed in the BL21star (DE3) strain of Escherichia coli using the pSUMO3 expression system and purified from the bacterial lysate using fast protein liquid chromatography, as previously described [21]. Purified samples of the protein were stored as 1 mg/ml solutions in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA at −80°C until they were used.
Protein Conformational Change Delayed by Steric Hindrance from an N-Linked Glycan
Preparation of zfPAI-1, rtPAI-1, and human PAI-1 To produce zfPAI-1 in E. coli, we cloned the coding sequence, without signal peptide, into the expression vector pET-47b(+) (Novagen), resulting in a construct with an N-terminal 6xHis-tag followed by an HRV 3C cleavage site (not utilized). The constructed plasmid was transformed into E. coli BL21(DE3)pLysS (Novagen) cells for protein expression. Cells were grown and induced at 25 °C and harvested 3 h later. The subsequent two-step purification procedure including HisTrap FF column followed by a Superdex 75 Prep Grade column were performed at 4 °C. The identity of the purified protein was verified by mass spectrometric peptide mapping analysis. For attempts at refolding, the zfPAI-1 produced in E. coli was denatured in either 8 M urea or 6 M guanidinium chloride and 0.1% β-mercaptoethanol. The denatured protein was rapidly diluted by dropwise addition to Functional Role of N-Linked Glycans 2873 150 ml of refolding buffer (50 mM Tris and 0.3 M NaCl, pH 8.5) while stirring gently in the cold room. After 1.5 h, the protein was concentrated by chromatography on a 1-ml HisTrap column. Approximately 40% of the initial protein amount was recovered after elution. Human PAI-1 was expressed in E. coli BL21(DE3) pLysS and purified as previously described.17 To produce glycosylated zfPAI-1, human PAI-1, or human PAI-1 D185N mutant, we cloned the full-length coding sequences into the mammalian expression vector pTT5.18 The D185N mutation in human PAI-1 was introduced by standard PCR methods and verified by DNA sequencing. The proteins were expressed by transient transfection of HEK293-6E cells growing in suspension with Mr ~ 25,000 linear polyethylenimine (Polysciences). Cells were grown in F17 serum-free medium (Invitrogen) supplemented with 0.1% Pluronic F-68 (Invitrogen), 4 mM L-Gln (Lonza), and 25 μg/ml G418 (Invitrogen). Twenty-four hours post-transfection, Tryptone N1 (Organotechnie SAS) was added to a final concentration of 0.5% (w/v). Approximately 96 h after transfection, conditioned medium was harvested, cleared by centrifugation, concentrated, and dialyzed. Purification was performed using Q Sepharose followed by size-exclusion chromatography on a Superdex 75 Prep Grade column. Glycosylated zfPAI-1 was refolded by dialysis into phosphate-buffered saline with 0.1% β-mercaptoethanol after treatment with 4 M guanidinium chloride and 0.1% β-mercaptoethanol.6 Purified zfPAI-1 from HEK293-6E was approximately 50% active. The active fraction was purified by affinity chromatography with a column of immobilized β-anhydrotrypsin.19 The so-called super-stable zfPAI-1 fraction was prepared by incubating active zfPAI-1 for 16 h at 37 °C, followed by purification on the β-anhydrotrypsin column. The flowthrough from the column was latent zfPAI-1. The active human PAI-1 was isolated by affinity chromatography on a β-anhydrotrypsin column. Latent human PAI-1 was produced by incubation for 20 h at 37 °C. Gel-filtration experiments on a Superdex 75 10/300 GL column ensured that the non- or deglycosylated zfPAI-1 was not aggregated. The coding sequence of rtPAI-1 without the signal peptide was cloned into the pET-47b(+) (Novagen) expression vector, resulting in a construct with an N-terminal 6xHis-tag. The construct was transformed intoE. coli BL21(DE3)pLysS (Novagen) cells for protein expression. The cells were grown and induced at 37 °C and harvested after 4 h. The protein purification was performed at 4 °C on a HisTrap FF column followed by a Superdex 75 Prep Grade column
Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1*
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 43, pp. 25795–25804, October 23, 2015
Expression and Purification of PAI-1 Mutant 14-1B—The recombinant PAI-1 mutant 14-1B (18) containing four point mutations (N150H, K154T, Q319L, and M354I), and a hexahistidine tag was expressed in Esherichia coli, using the expression vector pT7-PL and BL21 cells as previously described (19). In brief, cells were grown at 37 °C until mid-log phase followed by isopropyl-D-thiogalactoside induction at 16 °C overnight. Cells were harvested the next day and resuspended in buffer 20 mM MES, pH 6.1, 1 M NaCl, followed by ultracentrifugation. After sonication, cell pellets were separated by centrifugation, and the supernatant was loaded onto a 5-ml nickel affinity column (GE Healthcare). Subsequent Superdex75 gel filtration chromatography resulted in PAI-1 of greater than 95% purity
Protein Conformational Change Delayed by Steric Hindrance from an N-Linked Glycan
Preparation of zfPAI-1, rtPAI-1, and human PAI-1 To produce zfPAI-1 in E. coli, we cloned the coding sequence, without signal peptide, into the expression vector pET-47b(+) (Novagen), resulting in a construct with an N-terminal 6xHis-tag followed by an HRV 3C cleavage site (not utilized). The constructed plasmid was transformed into E. coli BL21(DE3)pLysS (Novagen) cells for protein expression. Cells were grown and induced at 25 °C and harvested 3 h later. The subsequent two-step purification procedure including HisTrap FF column followed by a Superdex 75 Prep Grade column were performed at 4 °C. The identity of the purified protein was verified by mass spectrometric peptide mapping analysis. For attempts at refolding, the zfPAI-1 produced in E. coli was denatured in either 8 M urea or 6 M guanidinium chloride and 0.1% β-mercaptoethanol. The denatured protein was rapidly diluted by dropwise addition to Functional Role of N-Linked Glycans 2873 150 ml of refolding buffer (50 mM Tris and 0.3 M NaCl, pH 8.5) while stirring gently in the cold room. After 1.5 h, the protein was concentrated by chromatography on a 1-ml HisTrap column. Approximately 40% of the initial protein amount was recovered after elution. Human PAI-1 was expressed in E. coli BL21(DE3) pLysS and purified as previously described.17 To produce glycosylated zfPAI-1, human PAI-1, or human PAI-1 D185N mutant, we cloned the full-length coding sequences into the mammalian expression vector pTT5.18 The D185N mutation in human PAI-1 was introduced by standard PCR methods and verified by DNA sequencing. The proteins were expressed by transient transfection of HEK293-6E cells growing in suspension with Mr ~ 25,000 linear polyethylenimine (Polysciences). Cells were grown in F17 serum-free medium (Invitrogen) supplemented with 0.1% Pluronic F-68 (Invitrogen), 4 mM L-Gln (Lonza), and 25 μg/ml G418 (Invitrogen). Twenty-four hours post-transfection, Tryptone N1 (Organotechnie SAS) was added to a final concentration of 0.5% (w/v). Approximately 96 h after transfection, conditioned medium was harvested, cleared by centrifugation, concentrated, and dialyzed. Purification was performed using Q Sepharose followed by size-exclusion chromatography on a Superdex 75 Prep Grade column. Glycosylated zfPAI-1 was refolded by dialysis into phosphate-buffered saline with 0.1% β-mercaptoethanol after treatment with 4 M guanidinium chloride and 0.1% β-mercaptoethanol.6 Purified zfPAI-1 from HEK293-6E was approximately 50% active. The active fraction was purified by affinity chromatography with a column of immobilized β-anhydrotrypsin.19 The so-called super-stable zfPAI-1 fraction was prepared by incubating active zfPAI-1 for 16 h at 37 °C, followed by purification on the β-anhydrotrypsin column. The flowthrough from the column was latent zfPAI-1. The active human PAI-1 was isolated by affinity chromatography on a β-anhydrotrypsin column. Latent human PAI-1 was produced by incubation for 20 h at 37 °C. Gel-filtration experiments on a Superdex 75 10/300 GL column ensured that the non- or deglycosylated zfPAI-1 was not aggregated. The coding sequence of rtPAI-1 without the signal peptide was cloned into the pET-47b(+) (Novagen) expression vector, resulting in a construct with an N-terminal 6xHis-tag. The construct was transformed intoE. coli BL21(DE3)pLysS (Novagen) cells for protein expression. The cells were grown and induced at 37 °C and harvested after 4 h. The protein purification was performed at 4 °C on a HisTrap FF column followed by a Superdex 75 Prep Grade column
Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1*
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 43, pp. 25795–25804, October 23, 2015
Expression and Purification of PAI-1 Mutant 14-1B—The recombinant PAI-1 mutant 14-1B (18) containing four point mutations (N150H, K154T, Q319L, and M354I), and a hexahistidine tag was expressed in Esherichia coli, using the expression vector pT7-PL and BL21 cells as previously described (19). In brief, cells were grown at 37 °C until mid-log phase followed by isopropyl-D-thiogalactoside induction at 16 °C overnight. Cells were harvested the next day and resuspended in buffer 20 mM MES, pH 6.1, 1 M NaCl, followed by ultracentrifugation. After sonication, cell pellets were separated by centrifugation, and the supernatant was loaded onto a 5-ml nickel affinity column (GE Healthcare). Subsequent Superdex75 gel filtration chromatography resulted in PAI-1 of greater than 95% purity
Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody
J. Biol. Chem. 2014 289: 23596
C1-INH and pKal orthologues from rat, cynomolgus monkey, and rabbit plasma were purified by Athens Research and Technology.
Protein expression and purification
To prevent disulfide-mediated aggregation, we prepared a cysteinefree
variant of crmA, which was found to retain normal inhibitory
activity against ICE ~data not shown!. This crmA variant was
expressed as inclusion bodies in Escherichia coli at 37 8C using the
pQE-60 expression system ~Qiagen, Hilden, Germany!. Inclusion
bodies were isolated, and the protein was refolded and purified as
previously described for a1PI ~Kwon et al., 1995!. The activity
was assayed by inhibition of the activity of ICE.
Cloning and Mutagenesis of Tengpin On the basis of the T. tengcongensis genomic sequence data (from the Beijing Genomic Institute), the gene for Tengpin was isolated using PCR and cloned into the E. coli expression vector pET-3a (Invitrogen). All single, double and triple mutants were introduced as previously described(Zheng et al., 2004). The primers for all mutants are shown in Supplementary Table 2. Positive clones were confirmed by DNA sequencing and transformed into RossettaBlue(DE3)pLysS Escherichia coli cells (Qiagen) for expression.
Expression and Purification For tengpin variants expression and purification, a freshly transformed colony was transferred into 1L 2YT broth (with 100 µg/ml of ampicillin, 34 µg/ml of chloromphenicol and 12.5 µg/ml of tetracycline), and grown overnight at 37 °C to saturation. This overnight culture was used to inoculate 10L 2YT broth (with antibiotics) and grown at 37 °C to an OD600 of around 0.7. The culture then was induced with 1mM IPTG for overnight at 16 °C. The cells were harvested, and pellet was resuspended in 200 ml of cold lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM DTT, 1mM EDTA, 1mM AEBSF) and broken by French-press. After centrifugation at 25,000g for 30min, polyethylenimine was added to a final concentration of 0.1% w/v), DNA binding protein was removed by centrifugation at 25,000g for 30min. Supernatant was dialysed against QA buffer (50 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM DTT, 1mM EDTA, 1 mM AEBSF) for overnight with 3 buffer changes, and then loaded onto Q-sepharose media. The column was washed with 10 column volumes of wash buffer (50mM Tris-HCl, pH 8.0, 50mM NaCl, 1mM DTT, 1mM EDTA, 1mM AEBSF), and then eluted with elution buffer (50mM TrisHCl, pH 8.0, 200mM NaCl, 1mM DTT, 1mM EDTA, 1mM AEBSF). The fractions containing the protein were pooled and extensively dialysised against QA buffer and then reloaded onto 5 ml HiTrap Q-sepharose column. The column was washed and the protein eluted using a 500 ml gradient applied into 50mM Tris-HCl, pH 8.0, 1M NaCl. Peak fractions were combined and dialysed, then (NH4)2SO4 was added to a final salt concentration of 1.7M. The high salt protein solution was loaded onto a pre-equilibrated (50mM Tris-HCl, pH 8.0, 1.7M (NH4)2SO4, 1mM DTT, 1mM EDTA, 1mM AEBSF) Hiload 26/10 phenyl-sepharose HP column, washed to baseline and eluted using a 250 ml linear gradient into 50mM Tris-HCl, pH 8.0.. Fractions corresponding to pure tengpin were combined, buffer exchanged into 50mM Tris-HCl, pH 8.0, 10mM NaCl and concentrated by eluted from 50 ml Q-sepharose column using 50mM Tris-HCl, pH 8.0, 300mM NaCl. In a final polishing step, protein was purified by superdex75 HR 10/30 column, and concentrated (Ultrafree-15 Centrifugal Filter Unit Amicon) to a final concentration of approximately 16mg ml -1 . The concentration of the recombinant mutated proteins was estimated by Bradford assay. N-terminal sequence confirmed the identity of tengpin
Cloning and Mutagenesis of Tengpin On the basis of the T. tengcongensis genomic sequence data (from the Beijing Genomic Institute), the gene for Tengpin was isolated using PCR and cloned into the E. coli expression vector pET-3a (Invitrogen). All single, double and triple mutants were introduced as previously described(Zheng et al., 2004). The primers for all mutants are shown in Supplementary Table 2. Positive clones were confirmed by DNA sequencing and transformed into RossettaBlue(DE3)pLysS Escherichia coli cells (Qiagen) for expression.
Expression and Purification For tengpin variants expression and purification, a freshly transformed colony was transferred into 1L 2YT broth (with 100 µg/ml of ampicillin, 34 µg/ml of chloromphenicol and 12.5 µg/ml of tetracycline), and grown overnight at 37 °C to saturation. This overnight culture was used to inoculate 10L 2YT broth (with antibiotics) and grown at 37 °C to an OD600 of around 0.7. The culture then was induced with 1mM IPTG for overnight at 16 °C. The cells were harvested, and pellet was resuspended in 200 ml of cold lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM DTT, 1mM EDTA, 1mM AEBSF) and broken by French-press. After centrifugation at 25,000g for 30min, polyethylenimine was added to a final concentration of 0.1% w/v), DNA binding protein was removed by centrifugation at 25,000g for 30min. Supernatant was dialysed against QA buffer (50 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM DTT, 1mM EDTA, 1 mM AEBSF) for overnight with 3 buffer changes, and then loaded onto Q-sepharose media. The column was washed with 10 column volumes of wash buffer (50mM Tris-HCl, pH 8.0, 50mM NaCl, 1mM DTT, 1mM EDTA, 1mM AEBSF), and then eluted with elution buffer (50mM TrisHCl, pH 8.0, 200mM NaCl, 1mM DTT, 1mM EDTA, 1mM AEBSF). The fractions containing the protein were pooled and extensively dialysised against QA buffer and then reloaded onto 5 ml HiTrap Q-sepharose column. The column was washed and the protein eluted using a 500 ml gradient applied into 50mM Tris-HCl, pH 8.0, 1M NaCl. Peak fractions were combined and dialysed, then (NH4)2SO4 was added to a final salt concentration of 1.7M. The high salt protein solution was loaded onto a pre-equilibrated (50mM Tris-HCl, pH 8.0, 1.7M (NH4)2SO4, 1mM DTT, 1mM EDTA, 1mM AEBSF) Hiload 26/10 phenyl-sepharose HP column, washed to baseline and eluted using a 250 ml linear gradient into 50mM Tris-HCl, pH 8.0.. Fractions corresponding to pure tengpin were combined, buffer exchanged into 50mM Tris-HCl, pH 8.0, 10mM NaCl and concentrated by eluted from 50 ml Q-sepharose column using 50mM Tris-HCl, pH 8.0, 300mM NaCl. In a final polishing step, protein was purified by superdex75 HR 10/30 column, and concentrated (Ultrafree-15 Centrifugal Filter Unit Amicon) to a final concentration of approximately 16mg ml -1 . The concentration of the recombinant mutated proteins was estimated by Bradford assay. N-terminal sequence confirmed the identity of tengpin
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