Earticle

Hyperacture rejection (HAR) of pig organs, upon xenotransplantation into primates, could partly be overcome by knocking out the alpha-Gal gene. However, xenotransplanted organs may still undergo immunological acture rejection (AR) or acute vascular rejection (AVR). Among several genes involved in AR and AVR, the hCD47 evades the monocyte/ macrophage mediated phagocytosis by identifying the self/non-self signal (CD47-SIRPa) whereas hTFPI participates in the regulation of coagulation pathway by acting upstream of the thrombin. In this study, we investigated hCD47 and hTFPI as two possible candidates for avoiding AR and AVR, respectively upon pig-to-human xenotransplantation. A co-expression vector for hCD47 and hTFPI was constructed using 2A peptides system (F2A) and transfected into the porcine kidney cell line (PK-15). The transfected cells stably expressed both hCD47 and hTFPI mRNA and proteins. Co-culture of non-transfected, hCD47-transfected, hTFPI-transfected or hCD47+hTPFI-transfected PK15 cells with natural killer (NK) cells, monocytes and macrophages confirmed the cytotoxic effect of hCD47 and revealed a synergistic effect of hCD47 and hTFPI co-transfection. There was an attractive survivability of 25～30% on each type of innate immune cell, NK cell and macrophage. These results suggest that transgenic pigs, genetically modified for hCD47 and hTFPI may be useful for overcoming xenograft rejection. Furthermore, cotransfection with hTFPI may enhance the cytotoxic effect of hCD47, possibly by assisting the hCD47-SIRPa binding by an unknown mechanism.

The chaperonin α-subunit gene (ApCpnA, 1,871 bp ORF) from the hyperthermophilic archaeon Aeropyrum pernix K1 was amplified by PCR and subcloned into vector pRSFDuet-1. The constructed pRSFDuet-ApCpnA (5.4 kb) was transformed into E. coli BL21 Rosetta (DE3). The E. coli transformant cell successfully expressed ApCpnA as 60.7 kDa protein in both soluble and insoluble fractions of cell lysate. The purified ApCpnA showed ATPase activity and its activity was dependent on temperature. When the alginate lyase gene (aly, 1.19 kb) from Pseudoalteromomas elyakovii was coexpressed with ApCpnA, alginate lyase was produced as a soluble and active form, speculating that ApCpnA facilitates the correct folding of alginate lyase. These results suggest that ApCpnA has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

LTB consisted of five identical polypeptides and, as a pentameric form, has been demonstrated to bind to the GM1 ganglioside at cellular surface. The recombinant LTB has attracted much attention due to its non-toxicity and potential as a strong immunogenic antigen and immuno adjuvant for both system and mucosal immune responses. Actinobacillus pleuropneumoniae is the causal agent of swine pleuropneumoniae, a disease resulting in morbidity and mortality of pigs and accordingly economic losses within the swine industry. Toxins (APX) produced by A. pleuropneumoniae appear to be important virulence factors of swine pleuropneumoniae and are thought to be of particular importance in the induction of protective immunity. In order to use S.cerevisiae for effective delivery of APX to the gut-associated lymphoid tissue, a chimeric APX fused with LTB subunit (LTB::APX) was constructed and tested for the oligimerization, which enables the chimeric complex to bind the intestinal membrane GM1-ganglioside receptor. Westhern blot ananlysis and ELISA indicated that a chimeric fusion protein was produced in recombinant S. cerevisiae. However, assembly into the native pentameric structure did not occur probably due to the stearic hindrance caused by the increased size of LTB::APX fusion construct. Therefore, co-expression strategy using episomal and integrative vectors for LTB and LTB::APX, respectively, was adapted for the oligomerization of LTB and LTB::APX fusion construct. GM1-ELISA indicated that the LTB::APX fusion construct, along with the LTB, was oligomerized to make the functional pentameric form. APX-specific antibody was observed when the recombinant yeast expressing both LTB as well as LTB::APX was orally administered. Antibody titer of orally administered mouse was enhanced when using the recombinant yeast expressing the pentameric form instead of recombinant yeast expressing either LTB::APX fusion only or APX alone. Better protection against challenge of A. pleuropneumoniae was also observed in the case of recombinant yeast compared to others. This study clearly indicated that the coexpression strategy enable the LTB::APX fusion construct to participate the pentameric formation, which resulted in facilitating the uptake of the recombinant protein through the GM1-ganglioside. Through which, an improved induction of systemic immune responses was achieved, which resulted in better protective response against the A. pleuropneumoniae challenge.

Hepatitis B virus causes the acute and chronic liver diseases. The gene coding for the S domain of hepatitis B virus surface antigen (sHBsAg) fused with the mating factor α (MFα) signal sequence was introduced by the chromosomal δ-integration to make S. cerevisiae 2805/pδMFα_sHBsAg strain. The sHBsAg gene was expressed under the control of the galactoseinducible GAL1 promoter. In this study, protein folding accessory proteins such as PDI1, SEC23, TRX2 and SCJ1 were coexpressed individually in recombinant S. cerevisiae 2805/pδMFα_sHBsAg to enhance the production of sHBsAg. Their coexpression led to a 2-fold increase each in the expression level of sHBsAg, compared to that of the control strain. The combinatorial expression of PDI1 and SEC23 gave a synergistic effect on the sHBsAg titer and productivity. Also KEX2 protease was coexpressed to obtain intact sHBsAg. In a glucose-limited fed-batch fermentation with YPDG medium, 15.2 g/L of dry cell mass and 5.81 mg/g cell of specific sHBsAg content were obtained in recombinant S. cerevisiae 2805/ pδMFα_sHBsAg +pPDI1+ pSEC23+pKEX2, which were approximately 11 and 12 times higher than those of the control strain, respectively.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4IgP)reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4IgM). However, limitations of hCTLA4IgP include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing b1,2-xylose and a1,3-fucose, which cause immunogenic reactions in humans. In the present study, human β1,4-galactose-extended hCTLA4IgP (hCTLA4IgP-Gal)was expressed through the coexpression of human β 1,4-galactosyltransferase (hGalT)and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4IgP-Galindicated that not only were the two glycans containing β1,4-galactose newly extended, but also glycans containing both b1,2-xylose and a1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4IgP,respectively. Unlike hCTLA4IgP, hCTLA4IgP-Gal was effective as an acceptor via β1,4-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4IgP-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4IgP, and the clearance pattern of hCTLA4IgP-Gal was close to that for hCTLA4IgM.These results indicate that the coexpression with hGalT and hCTLA4IgP is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.

Single chain Fv antibody (scFv) against c-Met is expected to be employed in clinical treatment or imaging of cancer cells due to the important biological roles of c-Met in the proliferation of malignancies. Hence it would be of importance to obtain a large amount of the recombinant anti-c-Met scFv for its practical utilization. Here, we show that the productivity of scFv against c-Met is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders, i.e. VL-linker-VH and VH-linker-VL, expressed them in the cytoplasm of E. coli trx/gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with VH/VL orientation was more than five times higher than that with VL/VH format. Coexpression of DsbC enhanced the yield of soluble amount of anti-c-Met scFv protein for both constructs. The scFv antibodies of the two different formats were purified through the interaction between Ni2+ and histidine tag on the c-terminus of recombinant protein, exhibiting almost same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with VH/VL or VL/VH formats and obtained similar results with the case of scFv antibodies.

The Dvl and Axin proteins, which are involved in the Wnt signaling pathway, each contain a conserved DIX domain in their sequences. The DIX domain mediates interaction between Dvl and Axin, which together play an important role in signal transduction. However, the extremely low production of DIX domain fragments in E. coli has prevented more widespread functional and structural studies. In this study, we demonstrate that the DIX domains of Dvl and Axin are expressed noticeably in a multi-cistronic system but not in a mono-cistronic system. Formation of the $DIX_{Dvl1}-DIX_{Axin1}$ complex was investigated by affinity chromatography, SEC and crystallization studies. Unstable DIX domains were stabilized by complexing with counterpart DIX domains. The results of the preliminary crystallization and diffraction of the $DIX_{Dvl1}-DIX_{Axin1}$ complex may prove useful for further crystallographic studies.

In an attempt to increase production of LK8, an 86-amino-acid kringle fragment of human apolipoprotein(a) with three disulfide linkages, protein disulfide isomerase (PDI) was coexpressed in recombinant Saccharomyces cerevisiae harboring the LK8 gene in the chromosome. Whereas overexpression of the LK8 gene without coexpressing PDI was detrimental to both host cell growth and LK8 production, coexpression of PDI increased the LK8 production level by 2.5-fold in batch cultivation and 5.0-fold in fed-batch cultivation compared with the control strain carrying only the genomic PDI gene.

Human heavy chain (H-) and light chain (L-) ferritins were amplified from a human cDNA library. Each ferritin gene was inserted downstream of the T7 promoter of bacterial expression vectors, and two types of coexpression vectors were constructed. The expression levels of recombinant ferritins ranged about 26-36% of whole-cell protein. H-ferritin exhibited a lower expression ratio compared with L-ferritin, by a coexpression system. However, the coexpression of HL-ferritins was significantly increased above the expression ratio of H-ferritin by cultivation without IPTG induction overnight. Purified recombinant H-, L-, HL-, and LH-ferritins were shown to be homo- and heteropolymeric high molecular complexes and it was indicated that their assembled subunits would be able to work functionally in the cell. Thus, these results indicate an improvement in the expression strategy of H-ferritin for heteropolymeric production and studies of ferritin assembly in Escherichia coli.

The endoxylanase (GenBank Access No. U51675) of Bacillus spp. and endoglucanase (GenBank Access No. AY466436) of Trichoderma spp. were separately inserted downstream of the yeast constitutive ADHI promoter, resulting in three different plasmids (pAGX1, pAGX2, and pAGX3) according to the transcription direction of two genes. When the yeast transformants, S. cerevisiae SEY2102 harboring each expression plasmid, were grown on YPD medium, the total activities of the enzymes were approximately 3.01 unit/ml, 3.24 unit/ml, and 7.56 unit/ml for endoxylanase and 0.60 unit/ml, 0.54 unit/ml, and 0.39 unit/ ml for endoglucanase, in the following order: the pAGX1, pAGX2, and pAGX3. More than 70% of the endoxylanase and endoglucanase activities was found in the extracellular media.

Baeyer-Villiger (BV) oxidation of cyclohexanone to ${\varepsilon}$-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum ${\varepsilon}$-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

Two genes, dps encoding decaprenyl diphosphate synthase and dxs encoding 1-deoxy-D-xylulose 5-phosphate synthase, were isolated from Rhizobium radiobacter ATCC 4718. DNA sequencing analysis of the dps and dxs genes revealed an open reading frame of 1,077 bp and 1,920 bp, respectively. The heterologous expression in Escherichia coli BL21(DE3) was carried out in order to identify their functions. Recombinant E. coli BL21(DE3) harboring the dps gene produced $CoQ_{10}$ as well as $CoQ_8$ and $CoQ_9$, whereas E. coli harboring only the dxs gene produced more $CoQ_8$ compared with the wild-type E. coli. Additionally, the coexpression of dps and dxs genes in E. coli was carried out. The recombinant E. coli harboring only the dps gene produced $0.21{\pm}0.04\;mg/l$ of $CoQ_{10}$, whereas the coexpressed E. coli with dps and dxs genes produced $0.37{\pm}0.07\;mg/l$ of $CoQ_{10}$. HPLC analysis also showed that the $CoQ_{10}$ fraction (100% of the total CoQs distribution) was increased from $15.86{\pm}0.66%$ (only dps) to $29.78{\pm}1.80%$ (dps and dxs).

Background: Both human leukocyte antigen (HLA) class I and programmed death-ligand 1 (PDL1) molecules are known to play important roles in cancer immunity. In this study, we evaluated HLA class I expression in resected adenocarcinoma of the lung, and investigated its prognostic impact in correlation with PD-L1 expression. Methods: HLA class I and PD-L1 expression was evaluated by immunohistochemistry in a total of 403 resected lung adenocarcinomas using tissue microarray. Correlations between the expression of HLA class I/PD-L1 and clinicopathologic features and prognostic significance were analyzed. Results: HLA class I expression was reduced in 91.6% of adenocarcinoma, and more frequently reduced in patients with younger age, absence of vascular invasion, and low pathologic stage (p = .033, p = .007, and p = .012, respectively). Positive PD-L1 expression in tumor cells was 16.1% (1% cut-off), and associated with poor differentiation, presence of vascular invasion and nodal metastasis (p < .001, p = .002, and p = .032, respectively). On survival analysis, HLA class I or PD-L1 expression alone did not show any statistical significance. On the integrated analysis, HLA class I (+)/PD-L1 (+) subgroup showed a significantly shorter overall survival than other groups (p = .001). Multivariate analysis revealed that coexpression of HLA class I and PD-L1 was an independent poor prognostic factor of lung adenocarcinoma. (p < .001; hazard ratio, 6.106; 95% confidence interval, 2.260 to 16.501). Conclusions: Lung adenocarcinoma with coexpression of HLA class I and PD-L1 was associated with poor prognosis. This subgroup may evade immune attack by expressing PD-L1 protein despite HLA expression.

Pseudoalteromonas elyakovii 유래 alginate lyase 유전자(aly)를 대장균에서 발현시켰을 때, 발현된 대부분의 유전자 산물은 내포체라는 불용성 응집체 형태로 생산되었다. Alginate lyase를 가용성 및 활성형으로 생산하기 위해 Aeropyrum pernix K1 유래의 초고온성 샤페로닌 ApCpnA와 ApCpnB를 공발현 파트너로 도입하였다. aly와 ApCpnA와 ApCpnB 각각과의 공발현 결과, aly 단독발현 때의 alginate lyase 활성 10.1 unit/g-soluble protein에서 ApCpnA와의 공발현 때는 83.1 unit/g-soluble protein, ApCpnB와의 공발현 때는 100.3 unit/g-soluble protein으로 가용성 및 활성형으로의 alginate lyase 생산이 8~10배 크게 향상되었다. 다양한 배양 조건들의 조사를 통해 alginate lyase 최대 생산을 위한 조건은 다음과 같았다: 1.0 mM IPTG, $25^{\circ}C$ 배양 온도(IPTG 유도 후), ApCpnB 공발현 파트너. 이러한 공발현 시스템은 대장균에서 기능적으로 또한 의학적으로 중요한 재조합 단백질의 산업적 생산에 크게 유용하게 사용될 것이다.

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At $25^{\circ}C$ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, $25^{\circ}C$ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

The irradiation of radioactive ${\gamma}-ray$ induces apoptosis of radiosensitive organs for homeostasis. In this study, we investigated the repair mechanisms for homeostasis in the small intestine after cell damage by $^{60}Co\;{\gamma}-ray$ irradiation. The apoptosis was most frequently observed in the crypt cells of the small intestine after four and six hours by radioactive ${\gamma}-ray$ irradiation, and the frequency of apoptosis was proportional to the amount of irradiation. Also, the number of apoptotic cells was coincident with expression pattern of p53. Interestingly, PCNA (proliferating cell nuclear antigen) which is engaged in DNA replication and repair was expressed in apoptotic cells of small intestinal crypts. Also, it was observed that cell-cycle regulator p21 which is known to induce cell-cycle arrest is co-expressed in the same apoptotic cells of irradiated small intestinal crypt cells. These findings suggest that the co-expression of PCNA and p21 proteins, which may lead to resistance to DNA damage through cell-cycle arrest is closely associated with repair of damaged gastrointestinal cells after ${\gamma}-ray$ irradiation.

서로 상관있는 유전자들의 발현조절이 질병이나 종양의 발생에 영향을 미치기 때문에 단일유전자 분석 대신 공통의 생물학적 요소를 지닌 유전자 집단 분석이 각광을 받게 되었고 생물학적으로 좀더 설명하기 쉬운 결과를 얻게 되었다. 표현형에 따라 유의한 차이를 보이는 유전자 집단을 찾는 여러 방법들이 있지만, 대부분의 방법들이 집단에 속한 유전자들의 표현형에 따른 발현의 차이를 탐색하거나 유전자들 사이의 공발현 구조가 다른지 탐색하는 것이다. 본 연구에서는 특이발현과 특이공발현의 차이를 모두 고려하는 탐색방법을 제시하였고 p53이란 유전자 자료와 모의자료를 이용하여 제시한 방법의 성능을 알아 보았다.

Gene set analysis utilizing biologic information is expected to produce more interpretable results because the occurrence of tumors (or diseases) is believed to be associated with the regulation of related genes. Many methods have been developed to identify statistically significant gene sets across different phenotypes; however, most focus exclusively on either the differential gene expression or the differential correlation structure in the gene set. This research provides a new method that simultaneously considers the differential expression of genes and differential coexpression with multiple genes in the gene set. Application of this NEW method is illustrated with real microarray data example, p53; subsequently, a simulation study compares its type I error rate and power with GSEA, SAMGS, GSCA and GSNCA.