Heparan sulfates targeting increases MHC class I- and MHC class II-restricted antigen presentation and CD8(+) T-cell response.

Heparan sulfates (HS) are carbohydrate moieties of HS proteoglycans (HSPGs). They often represent alternative attachment points for proteins or microorganisms targeting receptors. HSPGs, which are ubiquitously expressed, thereby participate in numerous biological processes.

Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase.

A single strategy to select RNA polymerase from bacteriophage T7 (T7 RNAP) mutants in Escherichia coli with enhanced thermostability or enzymatic activity is described. T7 RNAP has the ability to specifically transcribe genes under control of T7 phage promoter. By using random mutagenesis of the T7 RNAP gene in combination with an appropriate screening at 25 and 42°C, we have generated and selected E.

Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.

A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(L) genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21.

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.

BgK, a disulfide-containing sea anemone toxin blocking K+ channels, can be produced in Escherichia coli cytoplasm as a functional tagged protein.

BgK, a sea anemone peptide consisting of 37 amino acid residues and 3 disulfide bonds, blocks voltage-gated potassium (Kv1) channels. Here, we report a method for producing tagged BgK in Escherichia coli, as a soluble cytoplasmic protein. First, using peptidic synthesis, we show that addition of a 15 residue peptide (S.

Expression of recombinant alkaline phosphatase conjugates in Escherichia coli.

The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insertion of the DNA encoding the foreign peptides, proteic domains, or proteins between codons +6 and +7 of the phoa gene restores the initial frame of the phoa gene in the vector. Consequently, only recombinant clones appear as blue colonies when plating onto an agar medium containing a chromogenic substrate for PhoA.