Protein engineering, expression and purification

Bacterial strains and media

Cloning was performed using E. coli XL1-Blue competent cells grown in LB

medium, following established protocols.84 For heterologous overexpression, E. coli

BL21(DE3) or M15 pREP4 cells harbouring the appropriate expression construct were grown in PB medium (20 g l-1 tryptone, 10 g l-1 yeast extract, 5 g l-1glucose, 5 g l-1NaCl, 8.7 g l-1K2HPO4, pH 7.0). Kanamycin and ampicilin were used at 50 and 100 mg l-1, respectively.

Site-directed mutagenesis

Mutations were introduced using the Quick-Change Site-Directed Mutagenesis Kit (Stratagene). The following plasmids were used as templates for the PCR reactions: pQE-AppA5-125 for AppA,71 pRSET-ALBP for ALBP,30,85 pET22b(+) ∆98∆ IFABP

for ∆98∆.33

Following PCR reaction, the dammethylated plasmid templates were digested using the Dpn I restriction enzyme (Stratagene) and the vectors containing the new mutations were transformed into E. coli XL1-Blue and purified using QIAprep

Miniprep kit (Qiagen). All mutations were verified by sequencing (The Sequencing Service, Dundee, UK). The ALBP and AppA mutants verified by sequencing were transformed to E. coli M15-pREP4 for protein overexpression, and the ∆98∆

mutants were transformed toE. coliBL21(DE3). The mutagenic primers are specified in Tables 5-8.

Table 5Sequences of primers used o introduce mutations in AppA. Mutated bases are indicated in bold.

Primer Mutation acquired Sequence

Primer1 Primer 2

C19L/C20T TGCTGC/CTGACC

5’ GCTCGGATCTGGTTTCCCTGACCTACCGCAGCCTGGCGGC 3’ 5’ GCCGCCAGGCTGCGGTAGGTCAGGGAAACCAGATCCGAGC 3’ Primer 3 Primer 4 C110A TGC/GCG 5’ CACATGCAGCTCTCCGCGTCGGAGGCCGACATG 3’ 5’ CATGTCGGCCTCCGACGCGGAGAGCTGCATGTG 3’ Primer 5 Primer 6 Y21C TAC/TGC 5’ GTTTCCCTGACCTGCCGCAGCCTGGCG 3’ 5’ CGCCAGGCTGCGGCAGGTCAGGGAAAC 3’ Primer 7

Primer 8

Q63C CAG/TGC

5’ CAGGGCGTCTTCTTCTGCTGGCTCGAAGGCCAC 3’ 5’ GTGGCCTTCGAGCCAGCAGAAGAAGACGCCCTG 3’ Primer 9

Primer 10

W104C TGG/TGC

5’ CGCTTTGCGGGATGCCACATGCAGCTC 3’ 5’ GAGCTGCATGTGGCATCCCGCAAAGCG 3’ Primer 11 Primer 12 L65R CTC/CGT 5’ CTTCCAGTGGCGTGAAGGCCACC 3’ 5’ GGTGGCCTTCACGCCACTGGAAG 3’ Primer 13 Primer 14 V75R GTC/CGT 5’ CGTGGCGGAGCGTATGAGCCACATC 3’ 5’ GATGTGGCTCATACGCTCCGCCACG 3’

Table 6AppA mutants created by site-directed mutagenesis

AppA-BLUF domain mutant (Short) Name

AppA5-125C19L/C20T/Y21C/C110A AppAY21C

AppA5-125C19L/C20T/Q63C/C110A AppAQ63C

AppA5-125C19L/C20T/W104C/C110A AppAW104C

Table 7 Sequences for mutagenic primers used for C1A mutation in ALBP. Mutated bases are indicated

in bold.

Primer Mutation acquired Sequence

ALBP C1A-F ALBP C1A-R C1A TGT/GCC 5’ CCTCGAAGGTTTACAAAATGGCCGATGCCTTTGTGGGAACC 3’ 5’ GGTTCCCACAAAGGCATCGGCCATTTTGTAAACCTTCGAGG 3’

Table 8Sequences for mutagenic primers used for single point mutations in98. Mutated bases are

indicated in bold.

Primer Mutation acquired Sequence

Primer SC-F

Primer SC-R

Stop codon replaced with alanine TGA/GCC 5’ GAGTGGAGGCCAAGCGGGCCGGATCCGAATTCGAGC 3’ 5’ GCTCGAATTCGGATCCGGCCCGCTTGGCCTCCACTC 3’ Primer RG-F Primer RG-R G base deleted GCCGAT 5’ GCCAAGCGGGCCGATCCGAATTCGAG 3’ 5’ CTCGAATTCGGATCGGCCCGCTTGGC 3’

Primer 74 F Primer 74 R

K74Q AAG/CAG

5’ CGTGTAGACAATGGACAGGAGCTGATTGCTGTC 3’ 5’ GACAGCAATCAGCTCCTGTCCATTGTCTACACG 3’ Primer 104 F Primer 104 R K104Q AAG/CAG 5’ GCTCCGTCGACCAGCTTGCGGCCGC 3’ 5’ GCGGCCGCAAGCTGGTCGACGGAGC 3’ Cloning

To clone the ALBP gene into pQE30, cDNA corresponding to the ALBP C1A mutant was amplified by PCR using pRSET-ALBP C1A as template. The PCR product was purified from agarose gel (Wizard SV Gel and PCR Clean-Up System, Promega), digested with BamHI (Fermentas) and PstI (Fermentas) and ligated into BamH I/Pst I digested pQE30 vector (Qiagen) using T4 DNA ligase (Fermentas). The construct was verified by sequencing (The Sequencing Service, Dundee, UK).

Table 9Primers used for cloning of the ALBP C1A segment. Restriction sites (BamH I or Pst I) are underlined, the enterokinase site is given in italics.

Primer Sequence

Sc 1 5’-GCACGGATCCGATGACGATGACAAAATGGCCGATGCCTTTGTGG-3’ Sc 2 5’-GCACCTGCAG TCATGCCCTTTCATAAACTCTTG-3’

Protein expression and purification AppA

The AppA Y21C and AppA Q63C mutants were produced and purified according to a literature procedure.71 Immobilized nickel affinity chromatography was performed using a 5ml HisTrap™FF column (GE Healthcare Life Sciences).

ALBP

E. coli M15 pREP4 transformed with pQE-ALBP C1A was grown at 37°C in PB containing ampicillin and kanamycin. When the OD600reached 3, protein expression was induced by adding IPTG to a final concentration of 0.5 mM and growth was continued for 16 hours at 20°C. The cells were then harvested by centrifugation (4°C, 20 min, 3000 g) and resuspended in ice-cold lysis buffer (50 mM phosphate

buffer, 50 mM NaCl, 10% glycerol, 0.5% Nonidet P-40,86 pH 8.0) containing

protease inhibitor (Complete EDTA free, Roche). The cell-suspension was treated

with deoxyribonuclease (30 μg ml-1_{), ribonuclease (30 μg ml}-1

ml-1), held on ice for 30 min, sonicated (10 minutes with a 50% duty cycle) and centrifuged (4°C, 30 min, 27000 g). The soluble fraction was applied to a 5ml HisTrap™ FF column (GE Healthcare Life Sciences), the column was washed first with washing buffer (50 mM phosphate, 150 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) containing 0.5% detergent (Nonidet P-40),86 and then with an equivalent volume of washing buffer without detergent. The protein was eluted with elution buffer (50 mM phosphate, 150 mM NaCl, 500 mM imidazole, pH 8.0) and dialyzed overnight at 4ºC against 50 mM phosphate, 50 mM NaCl, pH 8.0 and stored at -20°C. Protein concentration was determined using the Bradford assay.68 Purity of the samples was checked by SDS-PAGE.

∆98∆

E. coli BL21(DE3), transformed with pET-22b(+)-∆98∆ IFABP was grown at 37°C

in PB containing ampicillin. When the OD600 reached 0.6, the protein expression was induced by adding IPTG (0.1 mM) and growth was continued for 16 hours at 25°C. The cells were then harvested by centrifugation (4°C, 20 min, 3000 g) and resuspended in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton- X 100, 15% glycerol, pH 8.0) containing protease inhibitors (Pefabloc, Roche). The

cell-suspension was treated with deoxyribonuclease (30 μg ml-1

), ribonuclease (30

μg ml-1

), and lysozyme (1 mg ml-1), held on ice for 30 min, sonicated (10 minutes with a 50% duty cycle) and centrifuged (4°C, 30 min, 27000 g). The soluble fraction was applied to a 5ml HisTrap™ FF column (GE Healthcare Life Sciences), the column was washed first with washing buffer (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) containing 0.5% detergent (Nonidet P-40),86 and then with an equal volume of the same washing buffer without detergent. The protein was eluted using elution buffer (50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0) and dialyzed at 4°C against 10 mM Tris-HCl, pH 8.0. After dialysis the protein was stored at -20°C, in buffer (10mM Tris-HCl, pH 8.0) containing 10% glycerol. Protein concentration was determined using the Bradford assay68 and the purity by SDS-PAGE.