Human liver-type glutaminase, catalytic domain

Target ID: GLS2A
Entry Clone ID: GL2A-s002
Allele ID: GLS2A-a007
Construct ID GLS2A-c007
Clone ID GLS2A-k007
Expression ID GLS2A-e039
Purification ID: GLS2A-p005
Entry clone source: from collaborator
Vector: pNIC28-Bsa4
E.coli strain: BL21(DE3)-R3-pRARE2
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag

Coding DNA sequence:

ATGCGCTCCATGAAGGCTCTGCAGAA
GGCCCTGAGCCGGGCTGGCAGTCACT
GCGGGCGAGGAGGCTGGGGTCACCCG
AGCCGGAGCCCCCTCCTTGGCGGGGG
CGTCCGGCACCACCTCAGTGAGGCCG
CGGCGCAGGGCAGAGAGACGCCACAC
AGCCACCAGCCGCAGCACCAGGATCA
TGATTCATCAGAAAGTGGCATGCTGT
CCCGCCTGGGTGATTTGCTCTTTTAC
ACTATTGCTGAAGGACAGGAACGAAT
CCCTATCCACAAGTTCACCACTGCAC
TAAAGGCCACTGGACTGCAGACATCA
GATCCTCGGCTCCGAGACTGCATGAG
CGAGATGCACCGCGTGGTCCAAGAGT
CCAGTAGTGGTGGCCTCTTGGACCGA
GATCTCTTCCGAAAGTGTGTGAGCAG
CAACATTGTGCTCCTGACCCAGGCAT
TCCGAAAGAAGTTTGTCATTCCTGAT
TTTGAGGAGTTCACGGGCCATGTGGA
TCGCATCTTTGAGGATGTCAAAGAGC
TCACTGGAGGCAAAGTGGCAGCCTAC
ATCCCTCAGCTGGCCAAGTCAAACCC
AGACCTGTGGGGTGTCTCCCTGTGCA
CTGTGGATGGTCAACGGCACTCTGTG
GGCCACACAAAGATCCCCTTCTGCCT
GCAGTCCTGTGTGAAGCCCCTCACCT
ATGCCATCTCCATAAGCACCCTAGGC
ACTGACTACGTGCACAAGTTTGTGGG
CAAAGAGCCAAGTGGCCTGCGCTACA
ACAAGCTCTCCCTCAATGAGGAAGGA
ATCCCCCATAACCCCATGGTCAATGC
TGGTGCCATTGTTGTCAGCTCCCTGA
TCAAGATGGACTGTAACAAAGCAGAG
AAGTTTGATTTTGTGTTGCAGTATCT
CAACAAAATGGCTGGGAATGAATACA
TGGGTTTCAGCAATGCCACATTCCAG
TCAGAGAAGGAAACAGGGGATCGGAA
TTATGCCATCGGCTATTATCTCAAGG
AAAAGAAGTGCTTTCCTAAGGGGGTG
GACATGATGGCTGCCCTTGATCTCTA
CTTCCAGCTGTGTTCTGTGGAGGTCA
CTTGTGAATCAGGCAGTGTCATGGCA
GCCACCCTCGCCAACGGTGGGATCTG
CCCCATCACAGGCGAGAGTGTGCTGA
GTGCTGAAGCAGTGCGCAACACCCTC
AGCCTCATGCATTCCTGCGGCATGTA
TGACTTCTCTGGCCAGTTTGCCTTCC
ACGTGGGCCTGCCAGCCAAGTCAGCT
GTATCAGGAGCCATCCTCCTGGTGGT
ACCCAATGTCATGGGAATGATGTGCC
TGTCACCCCCATTGGACAAGCTGGGG
AACAGCCATAGGGGGACCAGCTTCTG
CCAGAAGTTGGTGTCTCTCTTCAATT
TCCACAACTATGACAACCTGAGGCAC
TGTGCTCGGAAGTTAGACCCACGGCG
TGAAGGGGCAGAAATTCGGAACAAGA
CTGTGGTCAACCTGTTATTTGCTGCC
TATAGTGGCGATGTCTCAGCTCTTCG
AAGGTTTGCCTTGTCAGCCATGGATA
TGGAACAGAAAGACTATGACTCGCGC
ACAGCTCTGCATGTTGCTGCAGCTGA
AGGACACATCGAAGTTGTTAAATTCC
TGATCGAGGCTTGCAAAGTGAATCCT
TTTGCCAAGGACAGGTGGGGCAACAT
TCCCCTGGATGATGCTGTGCAGTTCA
ACCATCTGGAGGTGGTCAAACTGCTT
CAAGATTACCAGGACTCCTACACACT
CTCTGAAACTCAGGCTGAGGCAGCAG
CTGAGGCCCTGTCCAAAGAGAACTTA
GAAAGCATGGTATAG

Final protein sequence

mhhhhhhssgvdlgtenlyfq*sMIP
DFEEFTGHVDRIFEDVKELTGGKVAA
YIPQLAKSNPDLWGVSLCTVDGQRHS
VGHTKIPFCLQSCVKPLTYAISISTL
GTDYVHKFVGKEPSGLRYNKLSLNEE
GIPHNPMVNAGAIVVSSLIKMDCNKA
EKFDFVLQYLNKMAGNEYMGFSNATF
QSEKETGDRNYAIGYYLKEKKCFPKG
VDMMAALDLYFQLCSVEVTCESGSVM
AATLANGGICPITGESVLSAEAVRNT
LSLMHSCGMYDFSGQFAFHVGLPAKS
AVSGAILLVVPNVMGMMCLSPPLDKL
GNSHRGTSFCQKLVSLFNFHNYDNLR
HCARKLDPRREG

MHHHHHHSSGVDLGTENLYFQ*SM is the His6 tag (lower case) folowed by TEV protease recognition site (*).

Expression

Expression strain : BL21(DE3)-R3-pRARE2

Transformation

The construct DNA was transformed into competent cells of the expression strain by a standard heat shock procedure.

Glycerol stock preparation

One colony from the transformation was used to inoculate 1 ml of TB media containing 50 µg/ml kanamycin and 50 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.

Expression

A glycerol stock was used to inoculate 100 ml of TB media containing 50 µg/ml kanamycin and 50 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to inoculate 10L of TB media (7.5 ml starter culture used per 1L) containing 50 µg/ml kanamycin. When the OD600 reached approximately 0.8 the temperature was reduced to 18°C and after a further 1 hour the cells were induced by the addition of 0.1 mM IPTG. The expression was continued overnight.

Cell harvest

Cells were harvested by centrifugation at 6000 x g after which the supernatant was poured out and the cell pellet either placed in a -20°C freezer or used directly for purification.

Purification

Cell Lysis

Cell pellets were dissolved in approximately 50ml lysis buffer and broken by passing through a high pressure homogenizer at 15,000 psi for 4 cycles. The cell debris was pelleted at 35,000 x g and the supernatant used for further purification.

Lysis Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP, 1 tablet per 50 ml protease inhibitor cocktail EDTA-free (Roche)

Column 1 Ni-NTA affinity

The clarified cell extract was collected and added to 1.5 mL of Ni-NTA in a 250 mL plastic bottle and kept in cold room under rotation for 1h30min. The resin was then washed in a glass column with Binding Buffer (40 ml) and Wash Buffer (30 ml). The protein was eluted with 25 ml of Elution Buffer in 5 x 5 ml fractions. All of these steps were done in cold room.

Binding Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP

Wash Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.5 mM TCEP

Elution Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.5 mM TCEP

Column 2 Superdex 200 16/60 Gel Filtration

The first elution buffer fraction from column 1 was loaded in a Superdex 200 16/60 column (pre-equilibrated in GF Buffer) at 1.0 ml/min. 1.75 ml fractions were collected.

The protein was eluted at between 85 ml and 105 ml volume.

GF Buffer: 10 mM Hepes pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 5% Glycerol.

Column 3 HiTrap Q HP Ion Exchange

Pooled fractions from gel filtration (elution volume around 85-105 ml) were concentrated to 5 mL, and diluted with Zero Salt Buffer to a final NaCl concentration of 50 mM. The diluted sample was loaded onto the Hitrap Q HP anionic exchange column that was pre equilibrated with Low Salt Buffer. Elution was performed with a linear NaCl gradient from 0% to 25% of High Salt Buffer in 187.5 ml. 1.75 mL fractions were collected. Protein was typically eluted at around 200 mM NaCl.

Zero Salt Buffer: 25 mM HEPES pH 8.0, 0.5 mM TCEP, 5% Glycerol.

Low Salt Buffer: 25 mM HEPES pH 8.0, 50 mM NaCl, 0.5 mM TCEP, 5% Glycerol.

High Salt Buffer: 25 mM HEPES pH 8.0, 2 M NaCl, 0.5 mM TCEP, 5% Glycerol.

Concentration

The purified protein was concentrated to 13 mg/ml using Millipore 10k mwco concentrators.

Mass spectrometry characterization

Measured mass: 38425.14 Da

Expected mass: 38426.2 Da

Crystallization

Crystals were grown by the sitting drop vapour diffusion method at 20°C. A sitting drop consisting of 100 nl protein and 50 nl well solution was equilibrated against well solution containing 0.1M tris pH 8.5, 0.2 M sodium chloride and 25%(w/v) PEG 3350. Crystals were mounted in the presence of 25 % (v/v) ethylene glycol and flash-cooled in liquid nitrogen.

Data collection

Resolution: 2.2 Å

X-ray source: Diamond Light Source beamline IO3