Mass spectrometric evaluation of recombinant hemagglutinin structure conformations.
PBN-AR
Instytucja
Sieć Badawcza Łukasiewicz - Instytut Biotechnologii i Antybiotyków
Informacje podstawowe
Główny język publikacji
en
Czasopismo
PROTEIN SCIENCE
ISSN
0961-8368
EISSN
1469-896X
Wydawca
WILEY-BLACKWELL
DOI
URL
Rok publikacji
2015
Numer zeszytu
S1
Strony od-do
249
Numer tomu
24
Link do pełnego tekstu
Identyfikator DOI
Liczba arkuszy
Konferencja
Indeksowana w Scopus
nie
Indeksowana w Web of Science Core Collection
tak
Liczba cytowań z Web of Science Core Collection
Nazwa konferencji (skrócona)
29th Ann. Symp. of the Protein Society
Nazwa konferencji
29th Annual Symposium of the Protein Society
Początek konferencji
2015-07-22
Koniec konferencji
2015-07-25
Lokalizacja konferencji
Barcelona
Kraj konferencji
ES
Lista innych baz czasopism i abstraktów w których była indeksowana
Streszczenia
Język
en
Treść
Influenza virus (IV) hemagglutinin (HA) is a homotrimeric integral membrane glycoprotein that mediates receptor-binding and membrane fusion. It constitutes the prominent viral surface antigen and a main target for neutralizing antibodies. Bacterial, recombinant HA-based vaccines indicate high potential to confer protection against highly pathogenic (HP) avian IV (AIV) H5N1 and arise as alternative for the traditional egg- or cell culture-based manufacturing. Relatively short time of bacterial HAs production can be of great importance in case of a pandemic. Escherichia coli produced protein, based on the HA sequence of A/swan/Poland/305-135V08/2006(H5N1) HPAIV*, has been successfully expressed in the form of inclusion bodies at Institute of Biotechnology and Antibiotics. Refolded and purified antigen was obtained in a soluble form, isolated by reversed phase HPLC and identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/TOF MS). The performed research in a great extent allowed to confirm the amino acid sequence of the recombinant HA (rHA) assumed based on the cDNA and allowed to establish the location of a total of six disulfide bridges. However, during purification and storage of the rHA, apart from desired higher order rosette-like structures of the protein, other non-native species resulting from posttranslational modifications, misfolding, aggregation and degradation may occur what results in reduced vaccine potency. Here, besides the properly folded monomers, we indicate non-native aggregates induced by disulfide crosslinking. Moreover, several free cysteine residues and unexpected intrachain S-S were identified in rHA tryptic peptide maps. Cys 43 was found most susceptible to formation of disulfide bridges between the distinct chains of rHA. The above findings allow to assume that not all rHA particles fold to form the native structure. Reduced Cys residues exhibit tendency to undergo oxidation and uncontrolled S-S creation during storage. This may lead to activity drop and of non-native multimer formation. A covalent modification of several peptides of the rHA with a concomitant mass increase of 183 Da followed as a result of reaction with a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF).
Inne
System-identifier
PX-58c15afbd5dede0e300c9c67