Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli.

Bacterial lipoproteins are crucial antigens for protective immunity against bacterial pathogens. Expression of exogenous lipoproteins in Escherichia coli at high levels is thought to be an extremely difficult endeavor because it frequently results in incomplete or absent lipid modification. Previously, we identified a fusion sequence (D1) from a Neisseria meningitidis lipoprotein that induced a non-lipidated protein, E3 (the domain III of the dengue virus envelope protein), to become lipidated. However, without optimizing the growth conditions, some of the D1-fusion proteins were not lipidated. Here, we report the influence of medium components on the expression of recombinant lipoproteins in E. coli. For high-level expression of mature lipoproteins in the C43 (DE3) strain, M9 medium was better than M63 and the rich medium. Furthermore, we analyzed the influence of other media factors (including nitrogen and carbon sources, phosphate, ferrous ions, calcium, magnesium, and pH) on the levels of lipoprotein expression. The results showed that excess nitrogen sources and phosphate in M9 medium could increase the amount of immature lipoproteins, and glucose was a better carbon source than glycerol for expressing mature lipoproteins. We also found that lipoproteins tended to be completely processed in the alkaline environment, even in the nutrient-rich medium. Additional constructs expressing different immunogens or lipid signal peptides as targets were also utilized, demonstrating that these targets could be expressed as completely mature lipoproteins in the M9 medium but not in the rich medium. Our results provide the useful information for expressing mature exogenous lipoproteins in E. coli.

Genetic evidence for involvement of the alternative sigma factor SigI in controlling expression of the cell wall hydrolase gene lytE and contribution of LytE to heat survival of Bacillus subtilis.

The Bacillus subtilis cell wall hydrolase LytE is involved in cell wall turnover and cell separation during vegetative growth. lytE transcription is known to be driven by a YycF-activated SigA-dependent promoter. The cell wall regulator SigI is an alternative sigma factor that has been shown to be heat stress-inducible and to be essential for survival of B. subtilis at high temperature. However, none of the previously identified target genes of SigI contribute to heat resistance. We now demonstrate that lytE expression is heat-inducible and that heat induction of lytE expression is strongly dependent on SigI. We have also found that the lytE mutant shows the same growth defect at high temperature as the sigI mutant. Introducing an extra copy of lytE into the sigI mutant could rescue its growth defect. Our data strongly suggest that SigI-dependent lytE expression under heat stress is important for heat survival of B. subtilis.

Distinguishing features of a novel humanized anti-EGFR monoclonal antibody based on cetuximab with superior antitumor efficacy.

BACKGROUND: Cetuximab, the first approved EGFR targeting therapeutic antibody, is currently used to treat colorectal cancer and head and neck cancer. While effective, cetuximab is associated with a higher rate of skin rash, infusion reactions, and gastrointestinal toxicity, which was suggested to be linked to the presence of heterogeneous glycan contents on the Fab of the SP2/0-produced cetuximab. OBJECTIVE AND METHODS: To improve efficacy and minimize toxicity of EGFR inhibition treatment, we re-engineered cetuximab by humanizing its Fab regions and minimizing its glycan contents to generate HLX07. RESULTS: HLX07 binds to EGFR with similar affinity as cetuximab and shows better bioactivity compared to cetuximab in vitro. In vivo studies demonstrated that HLX07 significantly inhibited the growth of A431, FaDu, NCI-H292, and WiDr tumor cells and synergized them with chemotherapeutics and immune simulator agents such as anti-PD-1. In cynomolgus monkeys, 13-week repeat-dose GLP toxicokinetic studies showed minimal-to-mild toxicities in the dose range of up to 60 mg/kg/wk. In the preliminary phase 1 dose-escalation study, HLX07 had showed lower incidence of skin rashes with grade >2 severities. CONCLUSION: HLX07 is currently under phase 1/2 clinical development. We believe HLX07 would potentially be an alternative for patients who have been suffering from cetuximab-mediated toxicity.

Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy.

Cancer immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), represents a breakthrough in cancer treatment, resulting in unprecedented results in terms of overall and progression-free survival. Discovery and development of novel anti PD-1 inhibitors remains a field of intense investigation, where novel monoclonal antibodies (mAbs) and novel antibody formats (e.g., novel isotype, bispecific mAb and low-molecular-weight compounds) are major source of future therapeutic candidates. HLX10, a fully humanized IgG4 monoclonal antibody against PD-1 receptor, increased functional activities of human T-cells and showed in vitro, and anti-tumor activity in several tumor models. The combined inhibition of PD-1/PDL-1 and angiogenesis pathways using anti-VEGF antibody may enhance a sustained suppression of cancer-related angiogenesis and tumor elimination. To elucidate HLX10's mode of action, we solved the structure of HLX10 in complex with PD-1 receptor. Detailed epitope analysis showed that HLX10 has a unique mode of recognition compared to the clinically approved PD1 antibodies Pembrolizumab and Nivolumab. Notably, HLX10's epitope was closer to Pembrolizumab's epitope than Nivolumab's epitope. However, HLX10 and Pembrolizumab showed an opposite heavy chain (HC) and light chain (LC) usage, which recognizes several overlapping amino acid residues on PD-1. We compared HLX10 to Nivolumab and Pembrolizumab and it showed similar or better bioactivity in vitro and in vivo, providing a rationale for clinical evaluation in cancer immunotherapy.

Genetic evidence for the actin homolog gene mreBH and the bacitracin resistance gene bcrC as targets of the alternative sigma factor SigI of Bacillus subtilis.

The Bacillus subtilis sigI gene, which is a member of the class VI heat shock genes of the B. subtilis heat shock stimulon, encodes an alternative sigma factor whose regulon is poorly defined. In this study, by using a binary vector system, we showed that B. subtilis SigI could drive expression of a transcriptional fusion between the sigI regulatory region from Bacillus licheniformis, Bacillus sp. strain NRRL B-14911, B. subtilis, or Bacillus thuringiensis and the xylE reporter gene in B. subtilis. The transcriptional initiation sites of these fusions in B. subtilis were mapped by primer extension analyses. A putative consensus promoter sequence probably recognized by the B. subtilis SigI was thus deduced. Using a consensus sequence-based search procedure, we found putative sigmaI promoters preceding the actin homolog gene mreBH and the bacitracin resistance gene bcrC of B. subtilis. Overexpression of the B. subtilis sigI gene could specifically stimulate expression of both an mreBH promoter region-bgaB fusion and a bcrC promoter region-bgaB fusion. Expression of these two fusions at the amyE locus of the B. subtilis chromosome was heat inducible and SigI dependent as revealed by sigI gene disruption experiments. Primer extension analysis showed that the identified mreBH and bcrC transcriptional start sites were at appropriate distances from their sigmaI promoter elements. This further supports the notion that SigI can directly regulate mreBH and bcrC expression. Taken together, these results strongly suggest that mreBH and bcrC are new members of the SigI regulon.

The master transcription factor Spo0A is required for poly(3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis in Bacillus thuringiensis.

Bacillus thuringiensis is a gram-positive spore-forming bacterium that can accumulate poly(3-hydroxybutyrate) (PHB) as a carbon and energy storage substance in response to nutritional stress. The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events.

Identification and characterization of the Bacillus thuringiensis phaZ gene, encoding new intracellular poly-3-hydroxybutyrate depolymerase.

A gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome of Bacillus thuringiensis subsp. israelensis ATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designated phaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD of Pseudomonas putida was unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. The B. thuringiensis PhaZ was inactive against p-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-type B. thuringiensis cells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from a phaZ mutant lost this activity. The phaZ mutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S(102)-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate that B. thuringiensis harbors a new type of intracellular PHB depolymerase.