In an effect to broaden the application of the heat-inducible autolytic vector pUC18-cI857/p(R)-SRRz-rrnB previously developed, a new vector pUC18-cI857/p(R)(T41C)-SRRz-rrnB (pEAS-1b) was quantitatively characterized under various growth temperatures, heat induction temperatures and durations, and IPTG (isopropyl beta-d-thiogalactoside) induction times, after resolving its erratic lysis profile found previously. Escherichia coli BL21 cells harboring this vector grew well at temperatures <36 degrees C, and lysed efficiently (97.0 +/- 0.8%) just 0.5 h after heat induction at 42 degrees C for 30 min when cell growth was performed at 35 degrees C. Application of this autolytic vector either in 96-well plates, or on nitrocellulose membranes, or on agar plates led to facile, efficient and consistent release of intracellular recombinant enzymes (e.g., a lysis efficiency of 91.8 +/- 1.1% was obtained in 96-well plates). Further application in directed evolution was illustrated by improving the thermostability of amadoriase using this vector. This reagentless and in situ cell lysis method has the potentials for lysis of miniaturized samples in clinical diagnosis and bioanalytical detection, and even for lysis of cells in the microarray format.

The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify “hot spots” or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.

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BackgroundPeptides have recently become attractive for therapeutic applications. However, efficient production of medium- to large-sized peptides (30–100 amino acids [aa]) remains challenging both by recombinant and chemical synthesis. We previously reported the formation of active enzyme aggregates in Escherichia coli cells induced by the short β-structured peptide ELK16 (LELELKLKLELELKLK) and developed a streamlined protein expression and purification approach. In this approach, a cleavable self-aggregating tag (cSAT) consisting of an intein molecule and ELK16 was used to release the recombinant peptides with reasonable purity from active aggregates.ResultsIn this work, we extended the cSAT approach to a generalized expression and purification solution for a set of medium- to large-sized peptides with important therapeutic uses, including human glucagon-like peptide 1 (31 aa), B-type natriuretic peptide (32 aa), exendin 4 (39 aa), chemokine (C–C motif) ligand 5 (also known as RANTES, 66 aa), stromal cell-derived factor 1α (67 aa), insulin-like growth factor 1 (70 aa), and leptin (146 aa). After intein-mediated cleavage, the soluble peptides were released directly into the supernatant while insoluble peptides could be refolded and purified by reverse phase high-performance liquid chromatography. Additionally, an N-terminal thioredoxin tag was added upstream of the target peptides, which can be removed by enterokinase cleavage, generating native N-terminus for target peptides. Final yields of the peptides ranged from 0.1 to 1.8 μg/mg wet cell weight at laboratory scale.ConclusionsThe approach described in this study provides a fast and efficient route to express and purify peptides that are difficult or expensive to produce by chemical synthesis or by ordinary recombinant methods. It is particularly well suited for large peptides, peptides likely to be degraded, and peptides that have toxic effects on the host. It can greatly reduce the cost and time of downstream processing, and thus may be useful for both industrial manufacture and laboratory applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0534-3) contains supplementary material, which is available to authorized users.

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