Phox-homology domain of human sorting nexin 14

Target ID: SNX14B 
Entry Clone ID: SNX14B-s001
Allele ID: SNX14B-a101
Construct ID SNX14B-c101
Clone ID SNX14B-k101
Expression ID SNX14B-e110
Purification ID: SNX14B-p014
Entry clone source: MGC
Entry clone accession: BC005110  
Vector: pNIC28-Bsa4
E.coli strain: BL21(DE3)-R3-pRARE2 
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag

Coding DNA sequence

ATGGTGCCCTGGGTGCGGACGATGGG
GCAGAAGCTGAAGCAGCGGCTGCGAC
TGGACGTGGGACGCGAGATCTGCCGC
CAGTACCCGCTGTTCTGCTTCCTGCT
GCTCTGTCTCAGCGCCGCCTCCCTGC
TTCTTAACAGGTATATTCATATTTTA
ATGATCTTCTGGTCATTTGTTGCTGG
AGTTGTCACATTCTACTGCTCACTAG
GACCTGATTCTCTCTTACCAAATATA
TTCTTCACAATAAAATACAAACCCAA
GCAGTTAGGACTTCAGGAATTATTTC
CTCAAGGTCATAGCTGTGCTGTTTGT
GGTAAAGTGAAATGTAAACGACATAG
GCCTTCTTTGCTACTTGAAAACTACC
AGCCATGGCTAGACCTGAAAATTTCT
TCCAAGGTTGATGCATCTCTCTCAGA
GGTGGATATTCCATCTATTATAACCA
AGAAACTATTAAAAGCAGCAATGAAG
CATATAGAAGTGATAGTTAAAGCCAG
ACAGAAAGTAAAAAATACAGAGTTTT
TACAGCAAGCTGCTTTAGAAGAATAT
GGTCCAGAGCTTCATGTTGCTTTGAG
AAGTCGAAGAGATGAATTGCACTATT
TAAGGAAACTTACTGAACTGCTTTTT
CCTTATATTTTGCCTCCTAAAGCAAC
AGACTGCAGATCTCTGACCTTACTTA
TAAGAGAGATTCTGTCTGGCTCTGTG
TTCCTTCCTTCTTTGGATTTCCTAGC
TGATCCAGATACTGTGAATCATTTGC
TTATCATCTTCATAGATGACAGTCCA
CCTGAAAAAGCAACTGAACCGGCTTC
TCCTTTGGTTCCATTCTTGCAGAAAT
TTGCAGAACCTAGAAATAAAAAGCCA
TCTGTGCTGAAGTTAGAATTGAAGCA
AATCAGAGAGCAACAAGATCTTTTAT
TTCGTTTTATGAACTTTCTGAAACAA
GAAGGCGCAGTGCACGTGTTGCAGTT
TTGTTTGACTGTGGAGGAATTTAATG
ATAGAATTTTACGACCAGAATTATCA
AATGATGAAATGCTGTCTCTTCATGA
AGAATTGCAGAAGATTTATAAAACAT
ACTGTTTGGATGAAAGTATTGACAAA
ATTAGATTTGATCCCTTCATTGTAGA
AGAGATTCAAAGAATTGCTGAAGGCC
CATACATAGATGTTGTGAAACTTCAA
ACTATGAGATGTCTTTTTGAAGCATA
TGAACATGTTCTTTCCCTTTTGGAGA
ATGTATTTACTCCTATGTTCTGCCAT
AGTGATGAGTATTTCAGACAACTTTT
AAGAGGTGCAGAATCACCAACACGCA
ATTCAAAATTGAACAGGAACACACAG
AAAAGGGGAGAATCATTTGGAATCAG
CAGAATAGGTAGCAAAATTAAAGGAG
TATTCAAAAGTACCACAATGGAGGGA
GCTATGTTGCCTAATTATGGTGTAGC
TGAAGGTGAAGATGATTTTATTGAAG
AAGGTATTGTTGTAATGGAAGATGAT
TCTCCAGTGGAGGCTGTGAGCACACC
TAATACTCCCCGAAACCTTGCTGCAT
GGAAAATTAGCATTCCATATGTAGAC
TTTTTTGAGGATCCCTCCTCTGAAAG
GAAGGAGAAAAAAGAAAGAATTCCTG
TGTTTTGTATTGATGTTGAAAGAAAT
GATAGAAGAGCAGTTGGACACGAGCC
TGAACATTGGTCTGTCTATAGAAGAT
ATCTTGAATTCTATGTACTTGAATCA
AAACTAACAGAATTTCATGGTGCATT
TCCTGATGCCCAGCTTCCTTCTAAGA
GGATCATTGGCCCCAAAAATTATGAA
TTCTTAAAGTCAAAGAGGGAAGAGTT
CCAAGAATATCTACAGAAACTTCTGC
AGCATCCAGAACTGAGTAATAGTCAA
CTTCTGGCAGACTTTCTTTCCCCTAA
TGGTGGGGAAACACAATTTCTTGATA
AGATACTACCAGATGTAAATCTTGGG
AAAATTATAAAATCTGTTCCTGGAAA
ACTAATGAAAGAGAAAGGTCAGCATT
TGGAACCTTTTATCATGAATTTCATT
AATTCTTGTGAGTCTCCAAAGCCTAA
ACCAAGTAGACCAGAACTGACCATTC
TCAGCCCTACTTCAGAAAACAACAAG
AAGCTTTTCAATGATCTGTTTAAAAA
TAATGCAAACCGTGCTGAAAATACAG
AGAGAAAGCAAAATCAGAATTATTTT
ATGGAGGTGATGACTGTAGAAGGAGT
CTATGATTACCTGATGTATGTAGGAC
GGGTAGTTTTCCAGGTTCCTGACTGG
CTTCATCATCTCTTAATGGGAACTCG
AATCCTCTTTAAAAACACCCTGGAAA
TGTATACTGATTACTATCTTCAGTGT
AAACTAGAACAGCTATTTCAGGAGCA
CCGTTTGGTCTCACTCATAACACTTC
TCAGAGATGCTATATTCTGTGAAAAC
ACTGAACCTCGCTCTCTCCAAGATAA
GCAAAAAGGAGCAAAACAGACTTTTG
AAGAAATGATGAATTACATTCCAGAT
CTGTTAGTCAAGTGTATTGGTGAAGA
AACCAAGTATGAAAGCATCAGACTTC
TGTTTGATGGCTTACAGCAACCAGTA
CTCAACAAGCAGCTGACTTATGTTTT
ATTGGACATTGTGATACAGGAACTGT
TTCCAGAGCTCAATAAGGTACAAAAG
GAAGTTACCTCTGTGACATCTTGGAT
GTAA

Final protein sequence

mhhhhhhssgvdlgtenlyfq*sMTP
RNLAAWKISIPYVDFFEDPSSERKEK
KERIPVFCIDVERNDRRAVGHEPEHW
SVYRRYLEFYVLESKLTEFHGAFPDA
QLPSKRIIGPKNYEFLKSKREEFQEY
LQKLLQHPELSNSQLLADFLSPNGGE
TQFLDKILPDVNL

MHHHHHHSSGVDLGTENLYFQ*SM is the purification tag (lower case) plus TEV protease recognition site (*).

Expression

Expression strain

BL21(DE3)-R3-pRARE2 

A glycerol stock was used to inoculate 2X60 ml of TB media containing 50mg/ml kanamycin and 50 ìg/ml chloramphenicol, which was placed in a 37°C shaker overnight.  The next day this starter culture was used to inoculate 12L of TB media (10 ml starter culture used per 1L) containing 50 ìg/ml kanamycin.  When the OD600 reached approximately 1.0 the 

temperature was reduced to 18°C and after a further 30 minutes the cells were induced by the addition of 0.1 mM IPTG. 

Expression was continued overnight.

Cell harvest

Cells were harvested by centrifugation at 16,000 RPM after which the supernatant was poured out and the cell pellet either placed in a -80°C freezer or used directly for purification.

Purification

Buffers Used:

Binding/Lysis Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.01mM TCEP 

Wash Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.01mM TCEP 

Elution Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.01mM TCEP

Gel Filtration Buffer: 10 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 0.01mM TCEP

 

Cell Lysis

Cell pellets were dissolved in approximately 50ml lysis buffer and broken by passing through the homogeniser (x6) at a 

constant pressure of 15KPa.  The cell debris was pelleted at 16,000 RPM and the supernatant used for further purification.

Column 1

Ni-NTA (5.0 ml volume in a gravity-flow column).

The clarified cell extract was incubated with 5.0 ml pre-equilibriated 50% 

Ni_NTA bead solution for 1 hour at 4°C with rotation after which it was passed through a glass column. The column was then washed with 50ml Binding Buffer (2 x 25ml) and 50 ml Wash Buffer (2 x25 ml). The protein was eluted with 50 ml of 

Elution Buffer in 5 x 5 ml fractions. 

Column 2

Superdex s200 16/60 Gel Filtration. 

Elution fraction 1 and 2 were then pooled and concentrated to 5 ml (10 kDa mwco concentrator) and applied to the GF column (pre-equlibriated in GF buffer) at 1.0 ml/min. 1.0 ml fractions were collected.

Enzymatic treatment and purification 

The N-terminal His6- tag was cleaved by incubating overnight with TEV (20°C). Cleaved protein was purified by batch binding on 1ml pre-equilibriated 50% Ni-NTA bead solution. The column was then washed with 2x1ml Gel Filtration buffer, 2x1ml Wash buffer.

Concentration

To set up plates the sample was concentrated to 15.39 mg/ml using  a 10 kDa mwco concentrator.

Mass spectrometry characterisation

Expected  mass: 17373.7 Da 

Measured mass: 17361.1271  Da

Crystallization

Crystals were grown by vapour diffusion in sitting drop at 4°C. A sitting drop consisting of 50 nl protein and 100nl well solution was equilibrated against well solution containing 20%(w/v) PEG 3350 -- 0.2M sodium malonate.

Data collection 

Resolution:  2.6 Å  

X-ray source: Diamond Light Source beamline IO3