AviPure with two domain repeats was designed based on Staphylococcus aureus M0464 SpA B domain (accession number: KT377029) with some modification at the N, linker and C-terminal and the codon optimized sequence for expression in E. coli BL21 (DE3) was synthesized by Eurofingenomics (Erlangen, Germany) (Additional file 1: Figure S1). Molecular sub-cloning of AviPure into pET28a (+) was performed. Oligonucleotide primers, Avi-Fw ACT AGC TAG CGG ATC CCT GGC GGA TAA TAA ATT TAA C containing BamHI restriction site and Avi-Rv GAA TTC TGA AGC TTC CTT AGT GGC AAT GGC AAT G containing HindIII restriction site (underlined bases indicate BamHI and HindIII sites, respectively) also synthesized by Eurofingenomics (Erlangen, Germany) were used to extract the AviPure gene, while restriction enzymes BamHI-HF and HindIII-HF were purchased from New England Biolab (NEB, USA). The expression vector pET28a (+) was from Novagen (San Diego, CA, USA). AviPure PCR amplification was done using the Phusion® High-Fidelity DNA Polymerase (NEB, USA) consisted of 36 cycles of 94 °C for 30 s, 60 °C for 40 s and 72 °C for 2 min, and followed by a final extension step at 72 °C for 5 min, producing PCR gene product of about 407 bp. Plasmid pET28a (+) and PCR products were purified using NucleoSpin® Plasmid extraction and NucleoSpin® Gel and PCR Clean-up kit (Macherey–Nagel GmbH and Co. KG, Düren, Germany). The pET28a (+) and amplified AviPure gene product were individually digested (BamHI-HF and HindIII-HF) and ligated to obtain pAV01 with the N-terminal hexa-histidine tag that facilitate easy purification via Ni–NTA resin. The obtained expression vector was initially amplified in E. coli TOP10 cells and the correctness of the sequence was verified by analytical restriction digest and DNA sequencing (Eurofins MWG Operon, Ebersberg, Germany). The plasmid pAV01 was later isolated and subsequently transformed into E. coli BL21(DE3) cells to obtain the E. coli BL 21 (DE3):pAV01 for target protein expression.
Nucleotide sequence accession numbers
Gene sequences generated in this study were deposited in GenBank and the accession numbers are KT377029.
Five different media was analyzed for AviPure production: (1) Luria–Bertani medium broth (LB) (10 g/L bacto-tryptone, 5 g/L yeast extract, 5 g/L NaCl); (2) Terrific medium broth (TB) (12 g/L tryptone, 24 g/L yeast extract, 9.4 g/L K2HPO4, 2.2 g/L KH2PO4, 4 mL 98 % w/v glycerol); (3) M9 minimal medium (M9) (12.8 g/L Na2HPO2, 3 g/L KH2PO4, 0.5 g/L NaCl, 2 g/L NH4Cl, 20 g/L glucose, 0.01 g/L CaCl2, 0.12 g/L MgSO4, 0.002 g/L FeCl3); (4) Modified Minimal Medium (MMM) [30 g/L glucose, 1.2 g/L MgSO4 × 7H2O, 13.3 g/L KH2PO4, 4 g/L (NH4)2HPO4, 1.7 g/L citric acid, 10 mL of trace metals solution, 100 µL thiamine HCL (45 mg/mL)]; (5) Riesenberg Mineral Medium (RMM) (7.8 g/L KH2PO4, 2.33 g/L (NH4)2PO4, 10 g/L glucose, 0.5 g/L citric acid, 1.1 g/L MgSO4 × H2O, trace element solution, thiamine HCl.
E. coli BL 21 (DE3):pAV01 cells from a glycerol stock were initially plated on LB-agar plate containing appropriate antibiotics and incubated overnight at 37 °C, one single colony was later inoculated into 2 mL LB medium supplemented with appropriate antibiotics and cultured overnight at 37 °C with shaking at 225 rpm. The overnight culture then centrifuge and re-suspended in appropriate media, was transferred into Erlenmeyer flask containing 1 L cultures of each media and were incubated at 37 °C, at 225 rpm. All media were supplemented with 25 mg/L final amount of Kanamycin to avoid contaminations. Protein expression was induced by the addition of 1 mM final concentration Isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600 0.5. Samples were taken before and after induction and both the supernatant and the pellets were analyzed in a 15 % SDS-PAGE.
Mini-pilot scale fermentation of AviPure
For mini-pilot scale fermentation, TB and MMM were used. Initially, overnight cultures supplemented with appropriate antibiotics was prepared in 4 mL LB, further transferred and cultured to an OD600 of 1.0 in 500 mL Erlenmeyer flask of either TB or MMM media. The culture was later used to inoculate 9.5 L of TB and MMM media in a 30 L TV Techfors-S (Infors HT, Switzerland) fermenter previously sterilized at 121 °C for 20 min and cooled to 37 °C, respectively. During fermentation process some parameters were observed due to their importance; the change in OD, pH, aeration, antifoam, carbon source, agitation. At first the fermentation was run in batch mode with dissolved oxygen level maintained at required saturation (pO2) by using filtered air and with stirring speed in cascade mode in order to achieve and keep pO2 level constant. The production of foam was hindered by using 5 % antifoam solution (Sigma Aldrich, Germany) and the cultivation temperature was set at 37 °C. During fermentation, the pH of the media was maintained at 6.8 using a standard pH electrode (Mettler Toledo, USA) by the addition of phosphoric acid and liquid ammonia and monitored using the pH sensor unit. Before switching to fed-batch, the dissolved oxygen was used as a guide of the amount of feed needed (Yee and Blanch 1992), with the DO-stat used to balance the amount of glycerol/glucose needed at a time. Calibrated peristaltic pumps were used to control the feed rate for feed media (85 % glucose for MMM) which was determined by the metabolic rate of the culture. During fermentation, sampling of the culture in the medium was performed and analyzed for wet cell weight, and optical density at 600 nm. After 22 h, feed was reduced and protein expression was induced with 1 mM final concentration of IPTG solution. Cultivation was continued further 3 h till the end of cultivation time. E.coli cells pellets were collected by centrifugation using the Thermo Scientific Contifuge Stratos tabletop centrifuge, having a continuous flow rotor at 16,000 rpm, at 4 °C.
For AviPure purification, 5 g biomass (pellets) from the previously mini-pilot scale fermentation was re-suspended in 40 mL sonication/Wash buffer (50 mM Phosphate Buffer pH 7.5, 300 mM NaCl, 10 mM Imidazole) and 350 µL protease inhibitor cocktail mix. Direct sonication was performed for 45 min using amplitude of 90 % and 0.6 cycles; with the beaker containing cell suspension placed in ice/ice cold water and was continuously stirred to keep cool. After sonication, the lysate was centrifuged for 1 h at 16,000 rpm using a Beckman Coulot Avanti J-E centrifuge to get rid of cell debris. IMAC was used to purify the target protein and a 5 mL Ni–NTA column volume was chosen, equilibrated with sonication/wash buffer before loading. After centrifugation, the final 40 mL supernatant was loaded on the column. UPC–900 Amersham Biosciences AKTA FPLC system was employed for the purification. The elution buffer used was the same as the sonication/wash buffer, but contained a higher imidazole concentration (250 mM). The obtained protein was analyzed by SDS-PAGE following the method of Laemmli.
IgG affinity detection of AviPure via Western blot analysis
The affinity of the AviPure for IgG was investigated using protein affinity binding. Samples collected during cultivation (before and after induction) were resolved by a 15 % SDS-PAGE gel. Separated proteins were blotted onto nitrocellulose membranes (Whatman, Dassel, Germany) using a wet electro-blotting apparatus (Bio-Rad, USA). To prevent non-specific binding, the free binding sites of the membranes were saturated with 5 % fat free dry milk powder in TTBS (overnight at 4 °C with gentle agitation). Thereafter, the membrane was thoroughly washed five times, each for 5 min with 50 mL TTBS. After each incubation, the membrane was washed in the same way. The Goat anti-protein A polyclonal antibodies HRP conjugate at dilution of 1: 6000 was applied to the membrane, incubated for 1.5 h at room temperature with gentle agitation. This was followed by washing and the membranes were washed again and the proteins were subsequently visualized with a chemiluminescence kit (ECL) on X-ray film.
Ouchterlony radial immunodiffusion
To perform this method agar media was prepared by melting 2 g of agar in 50 mL buffer solution (50 mM PO4
−, 75 mM NaCl at pH 7.5) and diluting with 50 mL water. 450 μL of IgG were added to this solution. Petri dishes were used as plates for the test. Several holes were made in the solid agar solution to inject fractions and standards for comparison. Commercial protein A (Repligen, USA) (0.5 mg/mL) was used as a standard.
The purity and characterization of AviPure eluted from SEC was analyzed by mass spectrometry (Bruker Daltonik GmbH) (MALDI-TOF). 2, 5-dihydroxybenzoic acid (DHB) was applied as matrix solution and the Bruker PepMix Calibration Standard (# 222570, Bruker Daltonics) for the calibration of the instrument. For preparation of the matrix on the AnchorChip the DHB matrix layer method was used (for details see also AnchorChip Manual and Bruker Daltonics product sheets). The Autoflex II software was implemented for the evaluation of samples.