Red fluorescent protein eqFP611 and its genetically engineered dimeric variants

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four beta cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form.     

The effect of genetically engineered spider silk-dentin matrix protein 1 chimeric protein on hydroxyapatite nucleation

Spider silks exhibit remarkable mechanical properties while dentin matrix protein 1 provides controlled nucleation and hydroxyapatite growth. In the present work, these two attributes were combined via genetic engineering to form a chimera, a clone encoding consensus repeats from the major protein in the spider dragline silk of Nephila clavipes fused to the carboxyl terminal domain of dentin matrix protein 1 (CDMP1). The objective was to exploit the self-assembly and material properties of silk proteins with controlled hydroxyapatite (HA) formation from CDMP1, for novel biomaterial composites. The purified recombinant protein retained native-silk like self-assembly properties and beta-sheet structure when formed into films and treated with methanol. When the chimeric protein in solution was incubated with CaCl(2,) the secondary structure shifted from random coil to alpha-helix and beta-sheet, due to the interactions between the CDMP1 domain and Ca(2+). The control protein without the CDMP1 domain did not undergo a similar transition. Films formed from the recombinant protein were mineralized using simulated body fluids and induced the formation of calcium-deficient carbonated HA, Ca(10)(PO(4))(6)(OH)(2) based on SEM, EDS, FTIR and TEM analysis. This mineral phase was not formed on the films formed from the control spider silk protein without the CDMP1 domain. Considering the osteoconductivity of HA and the novel material features of spider silks, these new hybrid systems offer potential as biomaterials for a number of potential applications.     

Novel extracellular matrix for cell sheet recovery using genetically engineered elastin-like protein

Elastin-like peptides (ELPs) sequences are repeats of the pentapeptide GVGVP, and they have the ability to coaggregate reversibly, depending on the temperature. By exploiting this characteristic, a novel extracellular matrix protein (ECM) containing ELP was developed genetically to harvest a cell sheet from a culture dish. One of the ELP constructs, G288, consisted of 288 repeats of the sequence GVGVGP (G); it was attached to a hydrophobic dish surface. Next, cells with the sequence His-G36RG36, which has a His tag and an RGD sequence (R) that promotes attachment of the cell between the G36 sequences, consisted of 36 repeats of the sequence GVGVP, were added to the dish. After these cells became confluent, the temperature was changed to 20 degrees C in order to reverse the coaggregation. At this temperature, cells could be detached from the dish as a cell sheet. This genetically engineering method for construction of thermoresponsive ECM would be suitable to modify ECM with further functional domains.     

Skin genetically engineered as a bioreactor or a 'metabolic sink'

Genetically manipulated human keratinocytes can produce and secrete medically relevant proteins to the circulation. Genetically modified skin may also function as a 'metabolic sink' detoxifying the body of metabolites which accumulate in certain metabolic diseases. At the National Institutes of Health (NIH), Bethesda, Md., a clinical trial investigating the treatment of an ocular disease using the skin as a 'metabolic sink' for ornithine accumulating in gyrate atrophy patients is being prepared. The trial will involve the transplantation of a small patch of autologous keratinocytes, transduced ex vivo, onto the thighs of patients with gyrate atrophy. We are now investigating other diseases where this technology may be applicable such as in the treatment of hyperphenylalaninemia or hypercholesterolemia.     

Mucosal immunization with a genetically engineered pertussis toxin S1 fragment-cholera toxin subunit B chimeric protein

A chimeric protein consisting of a divalent pertussis toxin (PT) S1 fragment linked to the cholera toxin (Ctx) A(2)B fragment was constructed. The chimera induced a mucosal immunoglobulin A (IgA) and a serum IgG immune response to PT and CtxB in BALB/c mice following intranasal immunization. The immune sera neutralized PT in vitro. In the mouse model of Bordetella pertussis respiratory infection, the chimera-immunized animals showed a significant reduction in bacterial lung counts (P = 0.01) from that of the sham control group. Thus, a divalent S1 fragment CtxA2B chimera is an immunogenic antigen and can elicit a protective immunity.     

Mechanical properties and in vivo behavior of a biodegradable synthetic polymer microfiber-extracellular matrix hydrogel biohybrid scaffold

A biohybrid composite consisting of extracellular matrix (ECM) gel from porcine dermal tissue and biodegradable elastomeric fibers was generated and evaluated for soft tissue applications. ECM gel possesses attractive biocompatibility and bioactivity with weak mechanical properties and rapid degradation, while electrospun biodegradable poly(ester urethane)urea (PEUU) has good mechanical properties but limited cellular infiltration and tissue integration. A concurrent gel electrospray/polymer electrospinning method was employed to create ECM gel/PEUU fiber composites with attractive mechanical properties, including high flexibility and strength. Electron microscopy revealed a structure of interconnected fibrous layers embedded in ECM gel. Tensile mechanical properties could be tuned by altering the PEUU/ECM weight ratio. Scaffold tensile strengths for PEUU/ECM ratios of 67/33, 72/28 and 80/20 ranged from 80 to 187 kPa in the longitudinal axis (parallel to the collecting mandrel axis) and 41-91 kPa in the circumferential axis with 645-938% breaking strains. The 72/28 biohybrid composite and a control scaffold generated from electrospun PEUU alone were implanted into Lewis rats, replacing a full-thickness abdominal wall defect. At 4 wk, no infection or herniation was found at the implant site. Histological staining showed extensive cellular infiltration into the biohybrid scaffold with the newly developed tissue well integrated with the native periphery, while minimal cellular ingress into the electrospun PEUU scaffold was observed. Mechanical testing of explanted constructs showed evidence of substantial remodeling, with composite scaffolds adopting properties more comparable to the native abdominal wall. The described elastic biohybrid material imparts features of ECM gel bioactivity with PEUU strength and handling to provide a promising composite biomaterial for soft tissue repair and replacement.     

Characterization of genetically engineered mengoviruses in mice

We have shown that genetically engineered mengoviruses containing artificially shortened 5' noncoding poly(C) tracts (e.g., C0 or C13UC10) are dramatically attenuated in adult Swiss/ICR mice when compared to wild-type virus or to a genetically engineered virus containing a wild-type length poly(C) tract (C44UC10). To explore further the relationship between poly(C) tracts and virulence, we have conducted more extensive characterizations of several engineered viruses in the murine model. Both short and long poly(C) tract viruses were highly virulent in newborn mice, underscoring the importance of age in poly(C)-mediated attenuation. Virus vMC24, with a tract sequence of C13UC10, was as attenuated in 4-week-old BALB/c, C.C3-H2k/LiMcdJ, and DBA/2 mice as in Swiss/ICR mice. But it was more pathogenic for C57BL/6 mice, and highly virulent for C3H/Hej and C3H/Hen mice, demonstrating the importance of murine genotype. As expected from its virulence in all mouse strains, vMwt, with a poly(C) of C44UC10, induced higher levels of viremia than vMC24. The vMwt also induced higher levels of circulating interferon and had reduced pathogenicity in chemically immunosuppressed Swiss/ICR mice. Similar immunosuppression did not increase the virulence of vMC24. Collectively, the data suggest that endogenous immune components and the immune competence of the host play significant roles in determining the susceptibility of mice to mengovirus infection.     

Cell behavior on a CCN1 functionalized elastin-mimetic protein polymer

We report the design of an elastin-mimetic triblock copolymer with the ability to guide endothelial cell adhesion, spreading, and migration while maintaining the elastomeric properties of the protein polymer. The V2 ligand sequence from matricellular protein CCN1 (cysteine-rich 61, CYR61) was multimerized and cloned into elastin polymer LysB10, creating LysB10.V2. Cell adhesion studies demonstrated that a LysB10.V2 surface density of at least 40 pmol/cm² was required to elicit cell attachment. Peptide blocking studies confirmed V2 specific engagement with integrin receptor α_vβ₃ (P < 0.05) and we observed the formation of actin stress fiber networks and vinculin clustering, characteristic of focal adhesion assembly. Haptotatic migration assays demonstrated the ability of LysB10.V2 surfaces to stimulate migration of endothelial cells (P < 0.05). Significantly, we illustrated the ability of LysB10.V2 to support a quiescent endothelium. The CCN1 molecule functions to support many key biological processes necessary for tissue repair and thus presents a promising target for bioengineering applications. Collectively, our results demonstrate the potential to harness CCN1 specific function in the design of new scaffold materials for applications in regenerative medicine.     

基因工程双价抗体在肿瘤中的研究进展

基因工程抗体技术的发展加速了单链抗体的应用,但其稳定性差,亲和力低,功能单一,体内清除过快等特点影响了它的广泛应用。双价抗体作为一种新型小分子抗体,具有双价的结合位点,能够使抗原分子上的两个表位交联或使两个分子连接,可以模拟完整的单克隆抗体的抗原抗体反应,其构建方法有亮氨酸拉链法、利用部分抗体恒定区法、连接肽法、利用双聚化结构法、knobs into holes技术等,在乳腺癌、 直肠癌、淋巴瘤等的诊治方面均有很好的应用价值。     

Immunochemical analysis of a recombinant, genetically engineered, secreted HLA-A2/Q10b fusion protein.

We engineered a fusion gene which encodes the alpha 1 and alpha 2 domains of HLA-A2 with the alpha 3 and truncated transmembrane domains of the murine class I-like protein Q10b, and transferred it into mouse L cells along with the gene for human beta 2-microglobulin (beta 2m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was noncovalently associated with the human beta 2m light chain. Native detergent-solubilized HLA-A2 and secreted rA2/Q10b proteins were found to be similar by: (a) the binding to mouse monoclonal anti-HLA antibodies in an ELISA; (b) the blocking of lysis of HLA-A2+ cells by human anti-HLA-A2,-B17, anti-HLA-A2,9,28, and anti-HLA-A2,28 cross-reactive group (CREG) antisera in a complement-dependent cytotoxicity assay; and (c) the ability when coupled to Sepharose to selectively purify HLA-A2,9,28 and HLA-A2,28 CREG-specific antibodies. Mouse L cells expressing rA2/Q10b produced as much as 2.5 micrograms protein per 10(6) cells/day, or 50- to 100-fold more antigen on a per cell basis than the level of HLA-A2 expressed by B-lymphoblastoid cell line or spleen cells. Thus rA2/Q10b represents a viable alternative to detergent-solubilized HLA-A2 for purification of anti-HLA-A2 antibodies and analysis of anti-HLA-A2 immune responses.     

Secretion of heterologous proteins by genetically engineered Streptococcus gordonii

The secretion of heterologous proteins by Streptococcus gordonii from genes present on plasmids or on the chromosome has been achieved with a novel recombination system. We have constructed an integration-mediated transformation system in oral streptococci to clone foreign DNA into either resident plasmids or the host chromosome. In this system, Escherichia coli plasmid pACYC184 was extensively modified and utilized to construct anchor, integration, and heterodimer plasmids for introduction of the foreign genes. In order to evaluate this system, we cloned the gene for cycloisomaltooligosaccharide glucanotransferase (CITase) isolated from Bacillus circulans T3040 into S. gordonii. A portion of the CITase gene devoid of its signal sequence was fused inframe to the signal sequence of the Streptococcus sobrinus gtfI gene. The CITase fused gene was then introduced into either a resident plasmid or the S. gordonii chromosome. The presence of the enzymatically active CITase in the culture fluids from plasmid-borne transformants was confirmed. These results indicate that the integration-mediated transformation system described is an effective means of engineering recombinant streptococci capable of secreting heterologous proteins.     

Genetic synthesis and characterization of pH- and temperature-sensitive silk-elastinlike protein block copolymers

The purpose of this work was to synthesize and characterize a pH- and temperature-sensitive block copolymer containing repeating sequences from silk (Gly-Ala-Gly-Ala-Gly-Ser) and elastin (Gly-Val-Gly-Val-Pro) protein. The monomer contained one repeat of silk and eight repeat units of elastin, with the first valine in one of the elastin repeats being replaced by glutamic acid. The copolymer was synthesized using genetic engineering techniques. The sensitivity of the copolymer to pH and temperature was examined at various polymer concentrations and ionic strengths. Turbidity measurements were carried out over a temperature range of 20 to 100 degrees C at various pH, concentration, and ionic strength values. The introduction of an ionizable residue (glutamic acid) rendered the copolymer sensitive to changes in pH. The transition termperature (T(t)), the temperature at which the polymer became insoluble upon increase in temperature, was modulated by changing the pH. In general, the T(t) value, was found: (1) to increase with an increase in pH, (2) to decrease with increasing ionic strength, and (3) to decrease with increasing concentration. Results of these studies suggest that by strategic placement of charged amino acids in genetically engineered silk-elastinlike protein block copolymers it is possible to precisely control sensitivity to stimuli such as pH and temperature.     

Investigation of a genetically engineered mutant of barnacle troponin C containing a central helix deletion

To examine the importance of the central alpha-helix of troponin C (TnC) we have bacterially expressed one of the isoforms of barnacle TnC (BTnC2), BTnCWT, but without the aspartate residue at position 80 in the central helix (BTnC80-). This manipulation is expected to produce an approximately 100 degrees axial rotation of the C-domain with respect to the N-domain, and a net charge change of -1. BTnC80- mutant was able to restore force to TnC-depleted skinned barnacle myofibrillar bundles to a greater extent than wild-type protein (approximately = 170%). Competition experiments between BTnC80- and BTnC2-4-, a mutant lacking both of the calcium-specific sites (sites II and IV), shows that deletion of a single amino acid in the central helix results in a protein with increased affinity for the thin filament and one that is bound preferentially compared to BTnC2-4- when at equimolar concentrations.     

Expression of genetically engineered immunoconjugates of lymphotoxin and a chimeric anti-ganglioside GD2 antibody

Human lymphotoxin was genetically conjugated to the constant region of a human gamma 1 immunoglobulin gene at the end of either the second (CH2-LT) or third (CH3-LT) constant region domain. The altered heavy chain constant regions were combined in a plasmid vector together with the variable regions of a mouse anti-ganglioside GD2 antibody 14.18 and the human kappa constant region. The resulting immunoconjugate constructs were expressed in transfected hybridoma cells and tested for both their antibody and lymphotoxin activities. The two constructs were assembled to varying degrees depending on whether the third heavy chain constant region was present. Both forms retained their ability to bind antigen and mediate ADCC but only CH3-LT was able to mediate the lysis of melanoma target cells in the presence of human complement. Lymphotoxin activity, as defined in a cytolytic assay with mouse fibroblasts, was found to increase significantly as a function of heavy chain assembly and to be equivalent to unconjugated lymphotoxin. Neither of the constructs were cytotoxic for antigen-bearing melanoma cells that are normally resistant to lymphotoxin and tumor necrosis factor alpha. Such immunoconjugates may prove useful in targeting cytokines to the site of antigen-bearing cells in vivo. In this case, as a means of eliciting an inflammatory response at the site of a solid tumor.     

Secretion of genetically engineered human/mouse class I antigens

Two soluble, secreted forms of HLA-B7 were engineered by the creation of hybrid human/mouse molecules containing the polymorphic 5' region of the HLA-B7 gene and the secretory 3' region of the mouse Q10d gene. The hybrid, designated F1, is the first construct with only human extracytoplasmic domains, consisting of exons for the leader peptide and the three extracellular domains (alpha 1, alpha 2, alpha 3) of B7 spliced to the exons for the Q10d truncated transmembrane and 3' untranslated (3'UT) sequences. The second construct, designated C2, is similar but has the human alpha 3 replaced by the Q10 alpha 3 domain. Protein product from each construct was best demonstrated after gene transfection into the J27.2 cell line. In particular, secretion of the F1 product proves that the Q10 alpha 3 domain is not necessary for secretion of class I/Q10 hybrids. Moreover, the two soluble B7 forms, which differ only in their alpha 3 domain, are similarly recognized by monoclonal antibodies W6/32 (anti-HLA-ABC), BBM.1 (anti-human beta 2 microglobulin), and allo-B7-antibody, but differentially recognized by monoclonal antibody Q1/28 (anti-HLA class I heavy chain). Production of such soluble hybrid class I molecules in large amounts should allow critical structural and functional studies of these proteins.     

复制缺损型HBV表达显性阴性突变体抗HBV作用的研究

目的　探索HBV作为基因治疗载体的可能性及检验HBV点突变表达显性阴性突变体抗HBV的作用。方法　在表达完整HBV颗粒的质粒上 ,经基因修饰后分别表达核心 P蛋白及核心 表面抗原的融合蛋白 ,整合于具有HBV复制的 2 2 15细胞 ,形成细胞克隆 ,ELISA法检测细胞培养上清液中HBsAg和HBeAg ,斑点杂交法检测细胞内HBV核壳中HBVDNA ,PCR检测上清液中重组HBV颗粒。结果　 2 2 15 pMEP4组、2 2 15 CP组、2 2 15 CS组 ,对HBsAg平均抑制率分别为 2 74 %±3 83%、4 0 0 8%± 2 0 5 % (P <0 0 1)和 5 2 94 %± 1 93% (P <0 0 1) ;对HBeAg平均抑制率分别为4 4 6 %± 4 2 5 %、5 2 86 %± 1 32 % (P <0 0 1)和 4 1 6 0 %± 1 6 5 % (P <0 0 1) ;对HBV复制的抑制率分别为 15 3%、82 0 %和 6 7 2 %。仅在 2 2 15 CP组培养上清液中能检测出突变型HBV颗粒。结论　在细胞内表达显性阴性突变体具有干扰HBV复制及抑制HBV抗原表达的作用 ;经修饰后的HBV基因组在野生型HBV辅助下 ,仍能包装并分泌完整的HBV样颗粒。