Importance of Expression System in the Production of Unnatural Recombinant Proteins in Escherichia coli

Niraikulam Ayyadurai,Rameshkumar Neelamegam,Soundrarajan Nagasundarapandian,Selvakumar Edwardraja,Hyung Soon Park,Soo Jae Lee,Tae Hyeon Yoo,Hyungdon Yoon,이선구 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.3

In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/ MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/ MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system

다발성 골수종에서 p53 및 bcl-2 종양단백 발현

최종원,서진태 대한임상병리학회 1996 대한임상병리학회지 Vol.16 No.6

Hyper-Enhanced Production of Recombinant Proteins by the Partial Polyhedrin-fused Baculovirus Expression System

Sung Min Bae,Soo Dong Woo 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.10

The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Additionally, the several proteins expressed by the partial polyhedrin-fused expression system was markedly increased. However, we identified that hyper-expression of target protein varied depending on the partial polyhedrin. Therefore, we constructed the virus inducible partial polyhedrin fusion transient expression system. This system amenable for screening of suitable partial polyhedrin to produce the target protein. The present study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression

Chung, Jeong Min,Lee, Sangmin,Jung, Hyun Suk Elsevier 2017 Protein expression and purification Vol.133 No.-

<P><B>Abstract</B></P> <P>Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The expression and purification condition for truncated actin binding proteins was optimized from <I>Escherichia coli</I> strain. </LI> <LI> The treatment of sarkosyl-detergent increases the solubility of bacterial recombinant proteins. </LI> <LI> Minimizing concentration of sarkosyl was suggested for enhancing the production of soluble proteins. </LI> </UL> </P>

Expressed Protein Ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase: An Application to a Protein Expressed as an Inclusion Body

Kim, Hak-Jun,Shin, Hee-Jae,Kim, Hyun-Woo,Kang, Sung-Ho,Kim, Young-Tae Korean Chemical Society 2007 Bulletin of the Korean Chemical Society Vol.28 No.12

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

Differential expression of sex-related fat body proteins during the larval–pupal developmental stages of the silkworm (Bombyx mori)

Zhi-PingWu,Yan-Yan Liu,Guo-Qiang Chen,Ting-LiangWang,Jian-Zhong Tan 한국응용곤충학회 2014 Journal of Asia-Pacific Entomology Vol.17 No.1

The silkworm fat body is the site of many intermediary metabolic processes, and a source of sustenance forgrowth throughout the life cycle. Fat body proteins are responsible for storing nutrients, providing energy, andregulating hormones, and they have been identified using proteomic approaches. However, detailed differentialexpression of sex-related fat body proteins has not previously been evaluated. In the present study, we characterizedthe differential expression of sex-related fat body proteins, by using 2-dimensional gel electrophoresis(2-DE) followed bymass spectrometry identification and bioinformaticsmethods.We extracted the fat body proteinsfrom 5-day-old fifth instar larvae (L5), 10-day-old fifth instar larvae (corresponding to the end of spinning[LE]), and 0-day-old pupae (P0) of the multivoltine silkworm variety “Da Zao”. We confirmed the presence of 11important sex-specific expression proteins and 14 stage-specific expression proteins.We accurately identified 13of these specific expression proteins, including actin, calponin-like protein, 75 kDa subunit NADH, receptor foractivated protein kinase C from Bombyx mori (BmRACK), IMP (inosine monophosphate) cyclohydrolase, tropomyosin1, β-tubulin, hypothetical protein, antichymotrypsin precursor, and 30 K protein precursor.We showedthat BmRACK was differentially expressed betweenmale and female silkworms.Wediscuss the biological roles ofthe specific expression proteins during the larval–pupal developmental stages.

생체 외 도입에 따른 단백질의 세포 내 발현 양상

임현주,최종선,홍승희 대한구강악안면병리학회 2012 대한구강악안면병리학회지 Vol.36 No.6

Internalization and expression of extracellular molecules into cells and tissues is known very important process to biological processes and therapy of various diseases. In this study, we analyzed expression pattern of extracellular molecule after transduction into various human cells. To investigate cellular expression of internalized molecule, we used adenovirus containing green fluorescence protein. After infection of adenovirus into various human cells, the efficiency of intracellular gene expression was assessed with determining GFP expressing cells by fluorescence microscopy or FACS. After one day of adenovirus infection into HepG2 and A549, we observed that GFP expression was low at 10moi but expression levels were increased at 100moi in both cells. But, adenovirus infection into HCT116 showed low expression of GFP at concentrations from 1moi to 100moi. After 2 day infection with adenovirus, GFP expression level at 10moi and 100moi was highly increased in HepG2 and A549 compared with 1 day infection. Especially, GFP expression was significantly increased in HCT116after 2 days infection. However, GFP expressing SKOV3 cells by adenovirus infection were not found in all the experimental conditions tested. For quantitative analysis of GFP expression of cells by adenovirus infection, we carried out FACS analysis. As a result, GFP was expressed at very low levels at 1moi in all cells used in this experiment. GFP expression slightly increased after increasing moi to 10in HepG2, HCT116, and A549 cells. By 100moi infection of adenovirus, GFP expression was elevated to 10 fold higher than 10moi in HepG2 and A549 and about 4 fold elevation was observed in HCT116. A549 showed 20 fold higher expression of GFP than SKOV3. We also found that GFP expression by adenovirus infection was the highest in HepG2 cells. Protein expression was enhanced by increasing concentrations or time of adenovirus infection. In these results, GFP expression efficiency of adenoviral gene transduction reveals the highest in HepG2 and lowest in SKOV3 among the cells tested. Taken together, we could confirm that intracellular protein expression efficiency by transduction of extracellular gene was different in various human cells. Our study suggests that the cell types and cellular properties should be carefully examined to enhance expression efficiency of extracelluar molecules in biological research and disease therapy.

Original Article: Food Science/Microbiology : Development of Protein Biomarkers for the Authentication of Organic Rice

( Ju Young Lee ),( Jinkyu Lim ) 한국응용생명화학회 2015 Journal of Applied Biological Chemistry (J. Appl. Vol.58 No.4

The rice protein profiles of Oryza sativa L (Koshihikari) grown under organic and conventional cultivation regimes were compared on 2-D gels to develop diagnostic marker proteins for organic rice. The selected proteins, differentially expressed between organic and conventional rice, were compared with the differentially expressed proteins of another organic and conventional rice pairing, produced at a different location. In the first comparison among conventional, no-chemical, and organic rice grown in the same region, Korea, 13 proteins exhibiting differential expression in organic and conventionally grown plants were selected. Eight of the 13 proteins were down-regulated, and the 5 remaining proteins were up-regulated from conventional to organic rice. The second comparison pairing from Kyungju, revealed 12 differentially expressed proteins, with 8 down-regulated and 4 up-regulated proteins. Ten of the differentially expressed proteins that overlapped between the two comparison sets could not be clustered into any functional group using a functional annotation clustering tool. Further comparisons using another set of conventional and organic rice, belonging to a different variety of Oryza sativa L and produced in Sanchung, revealed 8 differentially expressed proteins, 5 of which were down-regulated and 3 of which were up-regulated in the organic rice. Overall, 3 differentially expressed proteins were commonly found in all three organic rice crops. These 3 proteins, along with other overlapping differentially expressed proteins, can provide a good starting point for the development of signature proteins that can be used for the authentication of organic rice with a follow-up studies with more comparison sets.

Proteomics를 이용한 등숙기 차이에 따른 콩 종실 저장단백질 발현양상 비교 분석

조성우,김태선,Swapan Kumar Roy,이철원,김홍식,우선희,권수정 한국작물학회 2015 한국작물학회지 Vol.60 No.1

In the present study, different expression ofprotein from Taekwang was revealed by 2-DE, and expressionsof protein on each week after flowering was investigated. After analysis of expression of protein, MALDI-TOF wasexecuted to identify expected protein function. Results revealedthat there were three patterns of expression of protein duringthe maturing. The first pattern was that proteins were graduallyexpressed as up-regulation from 1 week to 6 week. The secondpattern was that proteins were expressed gradually from 1 weekto 5 week and then it started down-regulation in 6 week. Thelast pattern was that proteins were gradually as up-regulationfrom 1 week to 3 week and then down-regulation until 6 week. This phenomenon suggests that young stage has more proteinrelated to correspondence mechanism against disease andgrowth and then maturing stage has more expression of proteinrelated to storage protein. In MALDI-TOF analysis, p24 oleosinisoform A protein was identified that relates oleosin whichis synthetic product in oil body. This protein spot increasedgradually until 5 week and then decreased after 5 week. Itexplained that the protein is active until maturing stage toprotect oil in seed and then its activity has gradually degraded. This result may be expected that a protein, related to growthof a seed has increased until maturing and then a seed fillsup with a storage protein. Keywords : Soybean, Seed Storage Protein, 2-DE, MALDI-TOF,Protein expression 본 연구는 국내 육성 품종인 태광콩의 등숙기에 따른 단백질 발현 양상을 비교함으로써 등숙기 단백질 발현의 차이에 대한 기초자료를 얻고자 수행하였다. 동한 개화 후 종실의 등숙이 진행됨에 따라서 단백질 발현 양상이 세가지 경향으로 나뉘어 지는 것을 확인하였다. 첫 번째는 등숙이 진행됨에 따라서 단백질 발현 정도가 증가하다가 감소되며,두 번째는 증가와 감소의 시기가 성숙기에 이루어지며, 세번째는 등숙기부터 성숙기까지 점진적으로 증가하는 것이다. 이러한 현상은 단백질의 기능에 따라 달라지는 것으로사료된다. 등숙 초기에는 등숙에 필요한 단백질의 발현이증가할 것이며 등숙 후기에는 저장단백질의 발현이 증가할것으로 사료된다. 따라서 향후 좀 더 많은 수의 단백질 spot들을 동정하여 어떤 기능을 가진 단백질이 등숙기에 따라단백질의 발현 양상이 달라지는지는 좀 더 면밀히 관찰할필요성이 있다고 사료된다.

대장암환자 조직에서의 p53 및 PCNA 단백질 발현

정석인,정구보 충북대학교 의과대학 충북대학교 의학연구소 1997 忠北醫大學術誌 Vol.7 No.2

본 실험은 85예의 대장암 조직에서 p53 단백질과 PCNA단백질의 발현을 면역조직화학 염색방법으로 관찰하여 다음과 같은 결과를 얻었다. p53 단백질의 발현율은 총 25예에서 양성은 11예(44%) 음성은 14예(56%)이었으며, 종양의 분화도가 나쁠수록, 종양의 위치가 5-결장직장 암에 위치한 경우, CEA가 5.0ng/dl 미만인 경우, Dukes씨 병기가 C인 경우, 임파절 전이수가 적을수록 발현율이 높은 경향을 보였다(p>0.05). 환자의 나이가 60세 이하인 경우 또는 종양의 크기가 커질수록 발현율이 높았으며 통계학적으로 유의한 차이가 있었다(p<0.05). PCNA 단백질의 labeling index는 평균70.48+18.83이었으며, 종양의 분화도가 나쁠수록, 종양의 위치가 5-결장직장에 위치한 경우, CEA치 5.0ng/dl 미만인 경우, Dukes씨 병기가 C인 경우, 환자의 나이가 적을수록, 임파절 전이수가 많을수록 labeling index가 높은 경향을 보였다(p>0.05). 병소의 크기가 5cm이상인 경우가 PCNA 단백질 labeling index가 높았으며, 통계학적으로 유의한 차이가 있었다(p<0.05). p53과 PCNA단백질의 발현 관계는 p53 단백질이 양성인 경우 양성율이 높은 때 PCNA의 발현율도 높아지는 경향을 보였다(p<0.10). 본 실험의 결과로 953 단백질은 대장암에서 환자의 나이 및 크기와 관련이 깊고, PCNA 단백질의 경우는 종양의 크기와 관련이 있는 것으로 보이나 이들의 발현이 대장암의 재발 및 예후에 어떠한 영향을 미치는가는 좀 더 연구해야 할 과제이다. Histopathologic staging of the colorectal cancer is known as the most important prognostic factor. But it is not enough to be used with full confidence in clinical field. It is the reason why various other prognostic factors have been intensively investigated. A few of them are p53 and PCNA protein. To evaluate the efficacy of p53 and PCNA protein expression as prognostic factors in the colorectal cancer, 25 specimens of colorectal cancer patients were selected. Immunohistochemical staining using ABC technique was performed for p53 and PCNA protein expression in paraffin tissue. The positive expression rate of p53 protein was 44% of specimen. p53 protein expression was not correlated with grade of differentiation, tumor site, Preoperative CEA, Dukes stage and number of metastatic lymph node. However, the expression rate of p53 protein was significantly higher in younger patients (under age 60) and in larger tumors (over 5cm) (p<0.05). The labeling index of PCNA protein was significantly higher in larger tumors (over 5cm) (p<0.05). PCNA protein expression was related to grade of differentiation, tumor site, preoperative CEA, Dukes stage, age of patients and number of metastatic lymph node. The over expression of p53 protein was correlated with labeling index of PCNA protein.