Biomedical subjects

Hua-Liang Huang

Publications and source records attributed to Hua-Liang Huang.

4 recordsLinked to original sources

PubMed2003

A new model of trispecific antibody resulting the cytotoxicity directed against tumor cells.

Bispecific antibodies (BsAb) with specificity to both tumor cells and CD3 molecule were believed to be promising immunological tools for the therapy with minimal residual diseases by activating cytotoxic T cells. However, without costimulatory molecule CD28, the activated T cells tended to apoptosis. In order to kill tumor cells more efficiently, a recombinant multifunctional single-chain trispecific antibody (scTsAb), which contains anti-ovarian carcinoma (OC) scFv, anti-CD3 scFv and VH domain of anti-CD28 antibody, was constructed and expressed in E. coli BL21 Star strain. The scTsAb showed strong binding avidities to membrane antigen of SK-OV-3 cell, CD3 molecule on Jurkat cell, and recombinant CD28 antigen. It was further demonstrated that this scTsAb could activate peripheral blood T cells to elicit strong cytotoxicity against SK-OV-3 cells. This new type of recombinant scFv antibody set up a new technological platform for T cells based immunotherapy against cancer, especially with the failure on MHC antigen presentation or absence of costimulating signal.

Antibodies, Bispecific↗

PubMed2002

Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli.

Single-chain Fv antibodies (scFv), a group of reconstructed molecules with several disulfide bonds, are prone to aggregate as inclusion bodies, the insoluble species of natural proteins, when expressed in Escherichia coli, especially at high level. Recovery of functionally active products from inclusion bodies is onerous and ineffective. We have increased the soluble and functional scFv yields by fusing either DsbC or DsbG, two E. coli disulfide isomerases with general chaperone function, to scFvs. Compared to the totally insoluble inclusion bodies of scFvs expressed separately, more than half of each fusion protein DsbC-scFv or DsbG-scFv was soluble, according to SDS-PAGE analysis. The more effective solubility was obtained when the fused protein DsbG-scFv was co-expressed simultaneously with DsbC under the same promoter. Under this condition, the soluble portion of DsbG-scFv increased from about 50% to 90% measured by scanning SDS-PAGE gel. Co-expression of DsbC can change fusion protein CBD-scFv from totally insoluble when expressed in E. coli separately to a considerable portion of soluble CBD-scFv. Antigen-binding activity assay showed that scFvs retained full affinity to specific antigens. We also determined that general molecular chaperones GroEL and GroES had no effects on the solubility of scFvs when co-expressed with scFv in E. coli. We propose that the correct formation of disulfide bonds in scFvs is the crucial factor responsible for solubility of scFvs.

Base Sequence↗

PubMed2002

A method for constructing reshaping single-domain antibody.

The aim of this research was to demonstrate a novel and practical method for constructing reshaping Single-domain antibodies. Different from other methods, our method does not need to model the configuration of antibodies with specific sequences to determine the sequences of human acceptor FRs and then determine which amino acid residues in human acceptor FRs should be substituted. Most importantly, reshaping and enhancing the antigen binding affinity shared one procedure at the same time. Using this method, the reshaping anti-CD28 single-domain antibodies were constructed. According to the amino acid sequence of a mouse anti-human CD28 monoclonal antibody VH, two most homologous sequences of human antibodies were selected from GenBank and one of them was used as a main framework region for constructing the reshaping antibody. Before the original mouse antibody CDRs were inserted into the human acceptor FRs, some amino acid residues which were different from those of the original mouse antibody in the corresponding positions of the human acceptor FRs were determined or alternatively mutated by their conservative properties in Kabat classification. When the synthesized nucleotide fragments in different length were spliced by overlap PCR into the entire reshaping genes, Taq DNA polymerase and high Mg2+ concentration were used to introduce more mutation in FRs and CDRs randomly. A phage library was constructed using these PCR products and several reshaping Single-domain antibodies with high antigen binding affinity were selected after three rounds of panning. Two of them were expressed in E. coli BL21 (DE3). The antigen-binding affinity of refolded proteins was still in a high level measured by ELISA. These results suggested that this method was feasible and efficient for constructing reshaping Single-domain antibodies.

Amino Acid Sequence↗

PubMed2002

[Escherichia coli disulfide-forming related proteins: structures, functions and their application in gene engineering for expressing heterologous proteins in Escherichia coli].

The formation of disulfide bonds in secreted proteins of E. coli is a synergetic process depending on a series of Dsb proteins containing DsbA, DsbB, DsbC, DsbD, DsbE and DsbG. DsbA functions as an oxidant to form a disulfide bond between two -SH- in vivo and DsbB reactivates DsbA by reoxidizing it. Both DsbC and DsbG, two periplasmic proteins with isomerase activity, can correct mis-paired disulfide bonds introduced by DsbA although they recognize different substrates. DsbD, an inner membrane protein, plays a role in reducing DsbC and DsbG in vivo. It is regarded that DsbE has the similar function with DsbD. All DsbA, DsbC and DsbG have chaperone activity besides involving in the formation of disulfide bonds. Furthermore, their chaperone activity can promote the formation of protein disulfide bonds. There are a few reports dealing with soluble expression of heterologous proteins containing disulfide bonds assisted by DsbA and DsbC in E. coli. So far there has been no reports about the soluble expression of heterologous proteins promoted by DsbG. Our experiments first demonstrated that both DsbC and DsbG can improve the expression of single chain antibodies as soluble and functional forms in E. coli, and DsbG has additive effects with DsbC.

Bacterial Proteins↗