SDS gels of FXAC and 23CF

fxac plicmbp  and 23cf nus purifications at the top

bottom gel 23CF pet 44 fplc peaks in the bottom

Fxac plicMBP TEV cleavage

peak fractions of Fxac plic MBP on S75 column

23cf purification of 15 Aug

23 CF  and FXAC pet32 and plicmbp pre and post induction

peak fplc practions 

purification of fxac plic 

Western blots of Fxac and 23CF

23cf pet44 fplc fractions 2nd peak shows the fusion protein 23cfNUS

fxac plic mbp before and after cleavage by TEV

fxac pet32 and fxac plic purification westerns

 23CF pet32 expression check

Fxac pet32 and fxac plic mbp expression check

  

FPLC PROFILES Fxac and 23CF various constructs

FXAC plicMBP DLS and FPLC 5sep

FXAC plic 4 litre purification 7 Sep

Lanes, ladder, lysate, beads after wash, elution1, elution2

Antigen5 pet32 purification

lanes page ruler unstained, lysate, pellet, Beads before wash, Beads afer wash, Elution1, Elution2, Flowthrough

FXAC mocr purification 4L 18 Sep

Prestained ladder, lys, pellet, BAW, elution1 , ....?

FXAC plicMBP Concentrated to 100 microlitres 9 Sep

23CFplicmbp purification 4L 22 Sep

lanes unstained ladder, lysate, pellet, BAW, Elution

Proti-Ace cleavage of FXAC plic concentrated to 1mg/ml

ladder, 4deg protein, ctrl 37 deg, alpha chymotrypsin, Elastase, Trypsin, papain, Endoproteinase Glu-C, Subtilisin

FXAC and 23 CF purification 12 Aug

lanes, ladder(prestained) lys23, lys fpet, lys fplic, 23 baw, fpet baw, fplic baw

ladder, 23baw2, FpetBAW2, fplic BAW2, 23E1, FpetE2, FpetE1, FplicE1, 23pellet, Fpet pellet

FXAC pet 32 and Fplic purification 19 August

lanes

ladder(prestained)  FXAC pet 32 lys, pellet, BAW , elution ----- FXAC plic lys, pellet, BAW, elution

23.5 kDa Culicine family protein purification 11 Sep 2016

23 CF pet32a purification
lanes Ladder, lysate, Beads after wash, Elution, FPLC peak

Induction check for fxac pet28, 23 pet 44, 23 mocr, fxac plic, 23 plic

gel 1 top = ladder , fxac pet28 pre, pet28 sumo pre, pet44 pre, 23pet44 pre, mocr pre , fxacpet28 post, pet28 sumo post, pet44 post,  23pet44 post

gel 2 (middle) = edem's pre, edem's post, ladder, zobia pre, zobia post, pmal crspi pre, pmal crspi post, 23 mocr pre, 23 mocr post, mocr post

gel3 (bottom)= plic mbp pre, plic mbp post,fxac plic pre, fxac plic post, ----, 23plic pre, 23plic post 

methods review of other serpins for protein expression

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.

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

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

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

FactorXa directed anticlotting serpin like protein - constructs available

FXAC pGEX6P1 Eco Xho
FXAC pet28SUMO Eco Xho
FXAC pet32a with thioredoxin tag - expression not good - Restriction sites - EcoRI and XhoI and precission site before start codon
FXAC plic MBP   - Expression good - TEV cleavage is not suitable
FXAC pet2OT with MOCR tag - Still in DH5 alpha
FXAC pet 44 with NUS tag
FXAC pet 28 - no expression - NcoI and XhoI

protein parameters for angiopoietin

ProtParam

User-provided sequence:

        10         20         30         40         50         60 
MNRQLWIIIF AILCVAQAEE DNPTTEKMEE LGIATINNFT REFYSYVEAV SQVLADLELT 

        70         80         90        100        110        120 
TTASITQIKH RIKHLLQEKC NLCSAKAEGP ALDQGYVTTS NGSVIPVSYE QTRFGGGWIV 

       130        140        150        160        170        180 
LMQRYDGTVR FNRSWAEYRD GFGMVGHEFW LGLERIHQMT KDAEYELMIE MQDFEGNYKY 

       190        200        210        220        230        240 
AGYDAFAVGP EEERYPLAKV GKFNKTAYVD SFGKHRGYGF STYDNDDNGC SNQYGRGGWW 

       250        260        270        280        290 
YYRKSCFGAS LTGIWQNKQD WKSISWVWFS TEKKQVPLKF ARMMMRLKTA E 

Number of amino acids: 291

Molecular weight: 33633.01

Theoretical pI: 6.03


Amino acid composition: 
Ala (A)  20   6.9%
Arg (R)  15   5.2%
Asn (N)  13   4.5%
Asp (D)  13   4.5%
Cys (C)   5   1.7%
Gln (Q)  14   4.8%
Glu (E)  23   7.9%
Gly (G)  25   8.6%
His (H)   5   1.7%
Ile (I)  15   5.2%
Leu (L)  18   6.2%
Lys (K)  18   6.2%
Met (M)  10   3.4%
Phe (F)  15   5.2%
Pro (P)   6   2.1%
Ser (S)  16   5.5%
Thr (T)  18   6.2%
Trp (W)  10   3.4%
Tyr (Y)  17   5.8%
Val (V)  15   5.2%
Pyl (O)   0   0.0%
Sec (U)   0   0.0%

 (B)   0   0.0%
 (Z)   0   0.0%
 (X)   0   0.0%

Total number of negatively charged residues (Asp + Glu): 36
Total number of positively charged residues (Arg + Lys): 33

Atomic composition:

Carbon      C       1513
Hydrogen    H       2273
Nitrogen    N        401
Oxygen      O        442
Sulfur      S         15

Formula: C1513H2273N401O442S15
Total number of atoms: 4644

Extinction coefficients:

Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.

Ext. coefficient    80580
Abs 0.1% (=1 g/l)   2.396, assuming all pairs of Cys residues form cystines


Ext. coefficient    80330
Abs 0.1% (=1 g/l)   2.388, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
                            >20 hours (yeast, in vivo).
                            >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 27.21
This classifies the protein as stable.



Aliphatic index: 66.05

Grand average of hydropathicity (GRAVY): -0.488

protein parameters for antigen 5

ProtParam

User-provided sequence:

        10         20         30         40         50         60 
MKSIISITIT VLAIICEGQA TNYCDPSLCA RGTPHIACNG LSTLSRTCGA GSFEVALNRA 

        70         80         90        100        110        120 
DQQLIVDLHN KLRSKVAMGQ QKNSAGQRFQ QACRMATLQW DPELAHIAAT NARRCVYGHD 

       130        140        150        160        170        180 
RCRNTASMKF AGQNIAIKYY YGMTFTNEQL LTGFINSWFS EFKDATPQQI GNVCSGCITG 

       190        200        210        220        230        240 
CNRVFPGLCN TAERVSNNPA RYPANYRGPA IGHFTQIVSD RTSRIGCSMV SYNKNGFINK 

       250 
LFVCNYGVTN IINQPVYVA 

Number of amino acids: 259

Molecular weight: 28479.55

Theoretical pI: 9.18


Amino acid composition: 
Ala (A)  24   9.3%
Arg (R)  16   6.2%
Asn (N)  22   8.5%
Asp (D)   7   2.7%
Cys (C)  14   5.4%
Gln (Q)  15   5.8%
Glu (E)   6   2.3%
Gly (G)  20   7.7%
His (H)   5   1.9%
Ile (I)  20   7.7%
Leu (L)  14   5.4%
Lys (K)   9   3.5%
Met (M)   6   2.3%
Phe (F)  11   4.2%
Pro (P)   9   3.5%
Ser (S)  17   6.6%
Thr (T)  18   6.9%
Trp (W)   2   0.8%
Tyr (Y)  10   3.9%
Val (V)  14   5.4%
Pyl (O)   0   0.0%
Sec (U)   0   0.0%

 (B)   0   0.0%
 (Z)   0   0.0%
 (X)   0   0.0%

Total number of negatively charged residues (Asp + Glu): 13
Total number of positively charged residues (Arg + Lys): 25

Atomic composition:

Carbon      C       1238
Hydrogen    H       1953
Nitrogen    N        365
Oxygen      O        368
Sulfur      S         20

Formula: C1238H1953N365O368S20
Total number of atoms: 3944

Extinction coefficients:

Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.

Ext. coefficient    26775
Abs 0.1% (=1 g/l)   0.940, assuming all pairs of Cys residues form cystines


Ext. coefficient    25900
Abs 0.1% (=1 g/l)   0.909, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
                            >20 hours (yeast, in vivo).
                            >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 30.75
This classifies the protein as stable.



Aliphatic index: 76.14

Grand average of hydropathicity (GRAVY): -0.151

PROTEIN PARAMETERS OF ANTIGEN5 MBP FUSION PROTEIN

MKSIISITIT VLAIICEGQA TNYCDPSLCA RGTPHIACNG LSTLSRTCGA GSFEVALNRA 

        70         80         90        100        110        120 
DQQLIVDLHN KLRSKVAMGQ QKNSAGQRFQ QACRMATLQW DPELAHIAAT NARRCVYGHD 

       130        140        150        160        170        180 
RCRNTASMKF AGQNIAIKYY YGMTFTNEQL LTGFINSWFS EFKDATPQQI GNVCSGCITG 

       190        200        210        220        230        240 
CNRVFPGLCN TAERVSNNPA RYPANYRGPA IGHFTQIVSD RTSRIGCSMV SYNKNGFINK 

       250        260        270        280        290        300 
LFVCNYGVTN IINQPVYVAM KIKTGARILA LSALTTMMFS ASALAKIEEG KLVIWINGDK 

       310        320        330        340        350        360 
GYNGLAEVGK KFEKDTGIKV TVEHPDKLEE KFPQVAATGD GPDIIFWAHD RFGGYAQSGL 

       370        380        390        400        410        420 
LAEITPDKAF QDKLYPFTWD AVRYNGKLIA YPIAVEALSL IYNKDLLPNP PKTWEEIPAL 

       430        440        450        460        470        480 
DKELKAKGKS ALMFNLQEPY FTWPLIAADG GYAFKYENGK YDIKDVGVDN AGAKAGLTFL 

       490        500        510        520        530        540 
VDLIKNKHMN ADTDYSIAEA AFNKGETAMT INGPWAWSNI DTSKVNYGVT VLPTFKGQPS 

       550        560        570        580        590        600 
KPFVGVLSAG INAASPNKEL AKEFLENYLL TDEGLEAVNK DKPLGAVALK SYEEELAKDP 

       610        620        630        640        650 
RIAATMENAQ KGEIMPNIPQ MSAFWYAVRT AVINAASGRQ TVDEALKDAQ TRITK 


Number of amino acids: 655

Molecular weight: 71849.18

Theoretical pI: 8.54


Amino acid composition: 
Ala (A)  74  11.3%
Arg (R)  23   3.5%
Asn (N)  43   6.6%
Asp (D)  31   4.7%
Cys (C)  14   2.1%
Gln (Q)  24   3.7%
Glu (E)  33   5.0%
Gly (G)  50   7.6%
His (H)   8   1.2%
Ile (I)  45   6.9%
Leu (L)  48   7.3%
Lys (K)  48   7.3%
Met (M)  15   2.3%
Phe (F)  27   4.1%
Pro (P)  30   4.6%
Ser (S)  32   4.9%
Thr (T)  41   6.3%
Trp (W)  10   1.5%
Tyr (Y)  25   3.8%
Val (V)  34   5.2%
Pyl (O)   0   0.0%
Sec (U)   0   0.0%

 (B)   0   0.0%
 (Z)   0   0.0%
 (X)   0   0.0%

Total number of negatively charged residues (Asp + Glu): 64
Total number of positively charged residues (Arg + Lys): 71

Atomic composition:

Carbon      C       3210
Hydrogen    H       5025
Nitrogen    N        865
Oxygen      O        949
Sulfur      S         29

Formula: C3210H5025N865O949S29
Total number of atoms: 10078

Extinction coefficients:

Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.

Ext. coefficient    93125
Abs 0.1% (=1 g/l)   1.296, assuming all pairs of Cys residues form cystines


Ext. coefficient    92250
Abs 0.1% (=1 g/l)   1.284, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
                            >20 hours (yeast, in vivo).
                            >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 23.20
This classifies the protein as stable.



Aliphatic index: 81.73

Grand average of hydropathicity (GRAVY): -0.212

protparam for srpn25

ProtParam

User-provided sequence:

        10         20         30         40         50         60 
MYLKVVILFS LQLVCYTQGG TPPSKPMAID YQAEFAWDLY KKLQLGFTQN LAIAPYSLRK 

        70         80         90        100        110        120 
IFVCLQQLTD SENPASAALS EQLKNVFKFN PKGKLPDLVR SRYSAQWTAL EREKGINTTT 

       130        140        150        160        170        180 
LAAVIGREKK TKLFRDLPNS CAIFAASLTP GSPKQMSRWF NAAMRNISKS GVQNFLSTSD 

       190        200        210        220        230        240 
IDRDWDFLVA DSWIFKGLWR YQFEEEHKTT CNFYTNSTSK GLMRFMYLQE YLKHGYFLEW 

       250        260        270        280        290        300 
NVQAVELPLH HGSSFSCMLM MPVKADIGVL IKSLNHRRFK DIYSKMSVSK TDVRLPQFTL 

       310        320        330        340        350        360 
RIKLSAKSIL QQFGFNAAFN ESVFHVFDNK NAVPLGDVIQ KVKLVMDHDG EQSAKMYVDR 

       370        380        390        400        410 
RMGNLFIAHQ QFIFVIFEKT QLVPIIVGHM VTASTPKDIG PESDEISCDK PPRY 

References and documentation are available.

Number of amino acids: 414

Molecular weight: 47392.91

Theoretical pI: 9.31


Amino acid composition: 
Ala (A)  27   6.5%
Arg (R)  18   4.3%
Asn (N)  18   4.3%
Asp (D)  21   5.1%
Cys (C)   6   1.4%
Gln (Q)  22   5.3%
Glu (E)  17   4.1%
Gly (G)  19   4.6%
His (H)   9   2.2%
Ile (I)  23   5.6%
Leu (L)  40   9.7%
Lys (K)  33   8.0%
Met (M)  14   3.4%
Phe (F)  28   6.8%
Pro (P)  19   4.6%
Ser (S)  33   8.0%
Thr (T)  20   4.8%
Trp (W)   7   1.7%
Tyr (Y)  14   3.4%
Val (V)  26   6.3%
Pyl (O)   0   0.0%
Sec (U)   0   0.0%

 (B)   0   0.0%
 (Z)   0   0.0%
 (X)   0   0.0%

Total number of negatively charged residues (Asp + Glu): 38
Total number of positively charged residues (Arg + Lys): 51

Atomic composition:

Carbon      C       2155
Hydrogen    H       3346
Nitrogen    N        566
Oxygen      O        598
Sulfur      S         20

Formula: C2155H3346N566O598S20
Total number of atoms: 6685

Extinction coefficients:

Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.

Ext. coefficient    59735
Abs 0.1% (=1 g/l)   1.260, assuming all pairs of Cys residues form cystines


Ext. coefficient    59360
Abs 0.1% (=1 g/l)   1.253, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
                            >20 hours (yeast, in vivo).
                            >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 40.09
This classifies the protein as unstable.



Aliphatic index: 84.08

Grand average of hydropathicity (GRAVY): -0.196

Protparam for MBP FXAC fusion

MKIKTGARIL ALSALTTMMF SASALAKIEE GKLVIWINGD KGYNGLAEVG KKFEKDTGIK 

        70         80         90        100        110        120 
VTVEHPDKLE EKFPQVAATG DGPDIIFWAH DRFGGYAQSG LLAEITPDKA FQDKLYPFTW 

       130        140        150        160        170        180 
DAVRYNGKLI AYPIAVEALS LIYNKDLLPN PPKTWEEIPA LDKELKAKGK SALMFNLQEP 

       190        200        210        220        230        240 
YFTWPLIAAD GGYAFKYENG KYDIKDVGVD NAGAKAGLTF LVDLIKNKHM NADTDYSIAE 

       250        260        270        280        290        300 
AAFNKGETAM TINGPWAWSN IDTSKVNYGV TVLPTFKGQP SKPFVGVLSA GINAASPNKE 

       310        320        330        340        350        360 
LAKEFLENYL LTDEGLEAVN KDKPLGAVAL KSYEEELAKD PRIAATMENA QKGEIMPNIP 

       370        380        390        400        410        420 
QMSAFWYAVR TAVINAASGR QTVDEALKDA QTRITKMYLK VVILFSLQLV CYTQGGTPPS 

       430        440        450        460        470        480 
KPMAIDYQAE FAWDLYKKLQ LGFTQNLAIA PYSLRKIFVC LQQLTDSENP ASAALSEQLK 

       490        500        510        520        530        540 
NVFKFNPKGK LPDLVRSRYS AQWTALEREK GINTTTLAAV IGREKKTKLF RDLPNSCAIF 

       550        560        570        580        590        600 
AASLTPGSPK QMSRWFNAAM RNISKSGVQN FLSTSDIDRD WDFLVADSWI FKGLWRYQFE 

       610        620        630        640        650        660 
EEHKTTCNFY TNSTSKGLMR FMYLQEYLKH GYFLEWNVQA VELPLHHGSS FSCMLMMPVK 

       670        680        690        700        710        720 
ADIGVLIKSL NHRRFKDIYS KMSVSKTDVR LPQFTLRIKL SAKSILQQFG FNAAFNESVF 

       730        740        750        760        770        780 
HVFDNKNAVP LGDVIQKVKL VMDHDGEQSA KMYVDRRMGN LFIAHQQFIF VIFEKTQLVP 

       790        800        810 
IIVGHMVTAS TPKDIGPESD EISCDKPPRY 

References and documentation are available.

Number of amino acids: 810

Molecular weight: 90762.53

Theoretical pI: 8.72


Amino acid composition: 
Ala (A)  77   9.5%
Arg (R)  25   3.1%
Asn (N)  39   4.8%
Asp (D)  45   5.6%
Cys (C)   6   0.7%
Gln (Q)  31   3.8%
Glu (E)  44   5.4%
Gly (G)  49   6.0%
His (H)  12   1.5%
Ile (I)  48   5.9%
Leu (L)  74   9.1%
Lys (K)  72   8.9%
Met (M)  23   2.8%
Phe (F)  44   5.4%
Pro (P)  40   4.9%
Ser (S)  48   5.9%
Thr (T)  43   5.3%
Trp (W)  15   1.9%
Tyr (Y)  29   3.6%
Val (V)  46   5.7%
Pyl (O)   0   0.0%
Sec (U)   0   0.0%

 (B)   0   0.0%
 (Z)   0   0.0%
 (X)   0   0.0%

Total number of negatively charged residues (Asp + Glu): 89
Total number of positively charged residues (Arg + Lys): 97

Atomic composition:

Carbon      C       4127
Hydrogen    H       6418
Nitrogen    N       1066
Oxygen      O       1179
Sulfur      S         29

Formula: C4127H6418N1066O1179S29
Total number of atoms: 12819

Extinction coefficients:

Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.

Ext. coefficient   126085
Abs 0.1% (=1 g/l)   1.389, assuming all pairs of Cys residues form cystines


Ext. coefficient   125710
Abs 0.1% (=1 g/l)   1.385, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
                            >20 hours (yeast, in vivo).
                            >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 29.82
This classifies the protein as stable.



Aliphatic index: 84.72

Grand average of hydropathicity (GRAVY): -0.223

ProtParam for 23.5 Culicine family protein

motif search - genome.jp

ProtParam

User-provided sequence:

        10         20         30         40         50         60 
MLQTIITLTV ILLPFSSSHN IPPWNGCYAV GCSTQQGCTP WNFYRYPCYR TCYYNRYCPP 

        70         80         90        100        110        120 
PSPPSKKCYK ITHVATGKSM TSSEVFNATT KAKYATVSSN PPANFLWTME PSYGGVNYIQ 

       130        140        150        160        170        180 
NANTLEFLKA DSNGTRLALN ETLDRSFQFR PIQTKEGASK LQLQSVFNNG FLFVQTLGQG 

       190        200 
GSTVQITTDP FTNYYQTIWC ITELYC 

Number of amino acids: 206

Molecular weight: 23202.35

Theoretical pI: 8.82


Amino acid composition: 
Ala (A)  10   4.9%
Arg (R)   6   2.9%
Asn (N)  15   7.3%
Asp (D)   3   1.5%
Cys (C)   9   4.4%
Gln (Q)  12   5.8%
Glu (E)   6   2.9%
Gly (G)  12   5.8%
His (H)   2   1.0%
Ile (I)  10   4.9%
Leu (L)  15   7.3%
Lys (K)   9   4.4%
Met (M)   3   1.5%
Phe (F)  11   5.3%
Pro (P)  15   7.3%
Ser (S)  17   8.3%
Thr (T)  24  11.7%
Trp (W)   4   1.9%
Tyr (Y)  14   6.8%
Val (V)   9   4.4%
Pyl (O)   0   0.0%
Sec (U)   0   0.0%

 (B)   0   0.0%
 (Z)   0   0.0%
 (X)   0   0.0%



Total number of negatively charged residues (Asp + Glu): 9
Total number of positively charged residues (Arg + Lys): 15

Atomic composition:

Carbon      C       1046
Hydrogen    H       1576
Nitrogen    N        268
Oxygen      O        307
Sulfur      S         12

Formula: C1046H1576N268O307S12
Total number of atoms: 3209

Extinction coefficients:

Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.

Ext. coefficient    43360
Abs 0.1% (=1 g/l)   1.869, assuming all pairs of Cys residues form cystines


Ext. coefficient    42860
Abs 0.1% (=1 g/l)   1.847, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
                            >20 hours (yeast, in vivo).
                            >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 43.45
This classifies the protein as unstable.



Aliphatic index: 64.85

Grand average of hydropathicity (GRAVY): -0.285

Protein Sequences

Protein Sequences

23.5kDa Culicine family protein

MLQTIITLTVILLPFSSSHNIPPWNGCYAVGCSTQQGCTPWNFYRYPCYRTCYYNRYCPPPSPPSKKCYKITHVATGKSMTSSEVFNATTKAKYATVSSNPPANFLWTMEPSYGGVNYIQNANTLEFLKADSNGTRLALNETLDRSFQFRPIQTKEGASKLQLQSVFNNGFLFVQTLGQGGSTVQITTDPFTNYYQTIWCITELYC

protein parameters
swiss model of 23 CF
swiss model closest homology paper - amaranthus agglutinin
swiss model closest homology paper 2- mushroom lectin polyporus
swiss model closest homology paper 3 - clostridium neurotoxin
swiss model closest homology paper 4 - mushroom lectin marasmius
original quote from genscript

SRPN25 

Factor Xa directed anti-clotting serpin like protein

MYLKVVILFSLQLVCYTQGGTPPSKPMAIDYQAEFAWDLYKKLQLGFTQNLAIAPYSLRKIFVCLQQLTDSENPASAALSEQLKNVFKFNPKGKLPDLVRSRYSAQWTALEREKGINTTTLAAVIGREKKTKLFRDLPNSCAIFAASLTPGSPKQMSRWFNAAMRNISKSGVQNFLSTSDIDRDWDFLVADSWIFKGLWRYQFEEEHKTTCNFYTNSTSKGLMRFMYLQEYLKHGYFLEWNVQAVELPLHHGSSFSCMLMMPVKADIGVLIKSLNHRRFKDIYSKMSVSKTDVRLPQFTLRIKLSAKSILQQFGFNAAFNESVFHVFDNKNAVPLGDVIQKVKLVMDHDGEQSAKMYVDRRMGNLFIAHQQFIFVIFEKTQLVPIIVGHMVTASTPKDIGPESDEISCDKPPRY

protein parameters

constructs available

PROTEIN BLAST FOR SERPIN25

Antigen 5

MKSIISITITVLAIICEGQATNYCDPSLCARGTPHIACNGLSTLSRTCGAGSFEVALNRADQQLIVDLHNKLRSKVAMGQQKNSAGQRFQQACRMATLQWDPELAHIAATNARRCVYGHDRCRNTASMKFAGQNIAIKYYYGMTFTNEQLLTGFINSWFSEFKDATPQQIGNVCSGCITGCNRVFPGLCNTAERVSNNPARYPANYRGPAIGHFTQIVSDRTSRIGCSMVSYNKNGFINKLFVCNYGVTNIINQPVYVA

Optimized nucleotide sequence (contains prescission protease site and ecoRI, XhoI sites)

GAATTCG
CTGGAAGTGCTGTTCCAGGGTCCGATGAAGAGCATCATTAGCATCACCATTACCGTGCTGGCGATCATTTGCGAA
GGCCAAGCGACCAACTATTGCGACCCGAGCCTGTGCGCGCGTGGTACCCCGCACATTGCGTGCAACGGCCTGAGC
ACCCTGAGCCGTACCTGCGGTGCGGGTAGCTTTGAGGTTGCGCTGAACCGTGCGGACCAGCAACTGATTGTGGAT
CTGCACAACAAGCTGCGTAGCAAAGTTGCGATGGGTCAGCAGAAGAACAGCGCGGGCCAGCGTTTTCAGCAAGCG
TGCCGTATGGCGACCCTGCAATGGGACCCGGAACTGGCGCACATTGCGGCGACCAACGCGCGTCGTTGCGTGTAT
GGTCACGATCGTTGCCGTAACACCGCGAGCATGAAGTTCGCGGGTCAGAACATCGCGATTAAATACTATTACGGC
ATGACCTTCACCAACGAGCAACTGCTGACCGGCTTTATCAACAGCTGGTTCAGCGAATTTAAGGACGCGACCCCG
CAGCAAATTGCGCGTTATCCGGCGAACTACCGTGGTCCGGCGATTGGTCACTTTACCCAGATTGTTAGCGATCGT
ACCAGCCGTATCGGTTGCAGCATGGTGAGCTACAACAAGAACGGCTTCATTAACAAACTGTTTGTGTGCAACTAT
GGTGTTACCAACATCATTAACCAGCCGGTGTACGTTGCGGGCAACGTGTGCAGCGGTTGCATCACCGGCTGCAAC
CGTGTTTTTCCGGGTCTGTGCAACACCGCGGAGCGTGTGAGCAACAACCCGTAA

CTCGAG

protein parameters for antigen 5

PROTEIN BLAST FOR ANTIGEN5 

TOP HOMOLOGY FOR ANTIGEN5 - ANT VENOM ALLERGEN

Angiopoietin

MNRQLWIIIFAILCVAQAEEDNPTTEKMEELGIATINNFTREFYSYVEAVSQVLADLELTTTASITQIKHRIKHLLQEKCNLCSAKAEGPALDQGYVTTSNGSVIPVSYEQTRFGGGWIVLMQRYDGTVRFNRSWAEYRDGFGMVGHEFWLGLERIHQMTKDAEYELMIEMQDFEGNYKYAGYDAFAVGPEEERYPLAKVGKFNKTAYVDSFGKHRGYGFSTYDNDDNGCSNQYGRGGWWYYRKSCFGASLTGIWQNKQDWKSISWVWFSTEKKQVPLKFARMMMRLKTAE

protein parameters for angiopoietin

Primer list

1)  antig5-for-pLICMBP  

5’ CCAGGGAGCAGCCTCG GAATTCG CTGGAAGTGCT  3’

wrong primer. out of frame restriction site

2) antig5-rev-pLICMBP

5’ GCAAAGCACCGGCCTCGTTA CTCGAGTTACGGGTTGTTGCTCAC 3’

3) antig5-Rev-pet2O-T  

5'  TTATCCACTTCCAATGTTATTA CTCGAGTTACGGGTTGTTGCTCAC 3'

wrong primer : do not include Restriction site CTCGAG after fusion tag

4) angio-rev-pet2OT

5'  TTATCCACTTCCAATGTTATTA CTCGAGTTACTCCGCGGTCTTCAG 3'

5)FXAC-For-pet2O-T  

  5'TACTTCCAATCCAATGCA ATGGCGATCGACTACCAGGC 3' 

6)FXAC-Rev-pet2O-T 

  5'TTATCCACTTCCAATGTTATTA TTAGGTCACCATGTGGCCAACAATG 3

7) 23.5CF-For-pet2O-T 

5'TACTTCCAATCCAATGCA GAATTCGCTGGAAGTTCT 3'

wrong primer :  do not include restriction site GAATTCG after fusion tag

8) 23.5CF-Rev-pet2O-T 

5'TTATCCACTTCCAATGTTATTA CTCGAGTTAGCAATACAGTTCGGTA 3' 

9) 23.5CF-for-pLICMBP  

5’ CCAGGGAGCAGCCTCG GAATTCGCTGGAAGTTCT  3’

wrong primer :  do not include restriction site GAATTCG after fusion tag

10) 23.5CF-rev-pLICMBP

5’ GCAAAGCACCGGCCTCGTTA CTCGAGTTAGCAATACAGTTCGGTA  3’

11) For Fxac pmalc5x NdeI

GATCGATC CAT ATG ATG GCG ATC GAC TAC CA