Engineered β\beta-lactoglobulin produced in E-coli : purification, biophysical and structural characterisation

Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant β-lactoglobulin such as CD spectra, ligand binding (n, K(a), ΔH, TΔS, ΔG), chemical and thermal stability (ΔG(D), C(mid)) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12033-016-9960-z) contains supplementary material, which is available to authorized users

Expression, purification and crystallization of bovine β lactoglobulin mutants L1AI2S and L1AI2S-V43N

β–Laktoglobulina (β-LgB) należy do rodziny lipokalin, białek mających zdolność wiązania i transportu bioaktywnych ligandów. Możliwa jest ich modyfikacja w celu zwiększenia powinowactwa i selektywności wiązania wybranych ligandów. Modyfikacje wprowadzane do struktury β LgB obejmowały mutację na N końcu (L1AI2S) ułatwiającą usuwanie N-końcowej metioniny oraz mutację w β-baryłce w pozycji 43 (L1AI2S-V43N), potencjalnie zwiększającą powinowactwo do ligandów z polarnymi grupami funkcyjnymi. Białko produkowano w bakteriach E.coli (Origami B oraz Lemo21) oraz izolowano z rozpuszczalnej i nierozpuszczalnej frakcji białek komórkowych (refolding). Białka oczyszczano używając technik chromatograficznych i wysalania oraz poddawano krystalizacji. Pomiary dyfraktometryczne prowadzono na dyfraktometrze SuperNova. Struktura krystaliczna wariantu L1AI2S izolowanego z frakcji rozpuszczalnej, wykazała zablokowane miejsce wiążące przez endogenny ligand. Otrzymane dwie struktury tego samego wariantu izolowanego z frakcji nierozpuszczalnej, wykazały puste wnętrze β-baryłki (możliwe tworzenie kompleksów z wybranymi ligandami). Pomiar widm CD wykazał zaburzenie procesu fałdowania białka z mutacją V43N. Poprawnie sfałdowany wariant L1AI2S-V43N można izolować z rozpuszczalnej frakcji białek w małych ilościach, z frakcji nierozpuszczalnej wariant ten produkowany jest w postaci częściowo zdenaturowanej.β-Lactoglobulin is a protein which belongs to lipocalin family. Lipocalins have ability to bind and transport bioactive ligands. To increase affinity and selectivity of ligand binding lipocalins can be re-engineered. N-terminal residues of bovine β-lactoglobulin were modified (L1AI2S) to facilitate cleavage of N-terminal methioninie. Valine 43 located inside β-barrel was replaced by asparagine (L1AI2S-V43N) to increase β-LgB affinity to ligands containing polar groups. Expression was carried out in E.coli strains Origami B and Lemo21. Both mutants were isolated from soluble and insoluble fractions of bacterial proteins. Proteins were purified by chromatography and salting out process. Obtained product was crystallized. X-ray diffraction data were collected using SuperNova diffractometer. Crystal structure of L1AI2S mutant isolated from soluble fraction, revealed binding site blocked by endogenous ligand. Two structures of the same mutant, isolated from insoluble fraction, proved that interior of β-barrel was unblocked (complexes of β-LgB with other ligands are possible). CD spectrum proved incorrect folding process of protein with V43N mutation. Properly folded L1AI2S-V43N mutant can be isolated from soluble fraction in small quantities. The same mutant is isolated in partially denaturated form from insoluble fraction

A Preliminary Study of FTIR Spectroscopy as a Potential Non-Invasive Screening Tool for Pediatric Precursor B Lymphoblastic Leukemia

Early detection of the most common pediatric neoplasm, B-cell precursor lymphoblastic leukemia (BCP-ALL), is challenging and requires invasive bone marrow biopsies. The purpose of this study was to establish new biomarkers for early screening to detect pediatric leukemia. In this small cohort study, Fourier transform infrared (FTIR) spectra were obtained from blood sera of 10 patients with BCP-ALL and were compared with the control samples from 10 children with some conditions other than neoplasm. Using various analytical approaches, including a new physical model, some significant differences were observable. The most important include: the different peak area ratio 2965/1645 cm−1 (p = 0.002); the lower average percentage of both β-sheet and β-turn protein structures in the sera of BCP-ALL patients (p = 0.03); an AdaBoost-based predictive model for classifying healthy vs. BCP-ALL patients with 85% accuracy; and the phase shift of the first derivative in the spectral range 1050–1042 cm−1 correlating with white blood cell (WBC) and blast cell count in BCP-ALL patients contrary to the samples obtained from healthy controls. Although verification in larger groups of patients will be necessary, these promising results suggest that FTIR spectroscopy may have future potential for the early screening of BCP-ALL