Glyco-engineering of yeast for production of therapeutic enzymes

오두병,Jin Young Gil,Jeong-Nam Park,Hoon Seo,Ohsuk Kwon,Seon Ah Cheon,Hyun Ah Kang 한국당과학회 2014 한국당과학회 학술대회 Vol.2014 No.1

Mannose-6-phosphate glycan for lysosomal targeting: various applications from enzyme replacement therapy to lysosome-targeting chimeras

서진호,오두병 한국통합생물학회 2022 Animal cells and systems Vol.26 No.3

A lysosome, an acidic membrane-bound organelle, contains hydrolytic enzymes to digest macromolecules for recycling. Many lysosomal enzymes (LEs) traffic to the lysosome through the mannose-6-phosphate (M6P)-dependent pathway. Some mannose residues of highmannose type N-glycans on LEs can be phosphorylated in the Golgi apparatus through twostep enzyme reactions. The consequent M6P moiety is recognized by M6P receptors (MPRs) on the trans-Golgi network membrane and delivered through the endo-lysosomal pathway. On the other hand, secreted LEs containing M6P glycans can be recaptured by MPRs on the plasma membrane and targeted to the lysosome. Enzyme replacement therapy (ERT) for lysosomal storage diseases exploits this M6P-MPR-dependent endocytosis to deliver recombinant enzymes to lysosomes. This review discusses various engineering and application technologies using M6P’s lysosomal targeting. Glyco-engineering for increasing M6P contents developed ‘Biobetter’ ERT enzymes with enhanced therapeutic efficacy. M6P-decorated peptides, proteins, liposomes, and nanoparticles have been developed for drug delivery and subcellular imaging. A recently developed lysosome-targeting chimera uses an M6P-based bifunctional binder to degrade specific extracellular and membrane proteins. The success and efficiency of M6P-based lysosomal targeting will boost further technological developments with new applications in the biomedical field.

의약용 당단백질에 부가된 당사슬의 중요성

김성훈,권오석,오두병,Kim, Seong-Hun,Kwon, Oh-Suk,Oh, Doo-Byoung 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.3

High value-added therapeutic proteins have been leading the biologics industry and occupied major portion of the market. More than 60% of the currently available protein therapeutics are glycoproteins attached with glycans which play crucial roles for the protein folding, therapeutic efficacy, in vivo half-life and immunogenecity. This review introduces the process of glycosylation and the impacts of glycans in the aspects of therapeutics. The important glycan structures in therapeutic performances were also summarized focusing on three representative categories of glycoproteins, cytokines, therapeutic antibody and enzyme. Currently, mammalian expression systems such as Chinese hamster ovary cells are preferred for the production of therapeutic glycoproteins due to their ability to synthesize glycans having similar structures with human type glycans. However, recent advances of plant glycoengineering to overcome the limitation originating from different glycan structures will soon allow to develop more efficient and economic plant-based production systems for therapeutic glycoproteins.

의약용 당단백질에 부가된 당사슬의 중요성

김성훈,권오석,오두병 한국식물생명공학회 2010 JOURNAL OF PLANT BIOTECHNOLOGY Vol.37 No.3

High value-added therapeutic proteins have been leading the biologics industry and occupied major portion of the market. More than 60% of the currently available protein therapeutics are glycoproteins attached with glycans which play crucial roles for the protein folding, therapeutic efficacy,in vivo half-life and immunogenecity. This review introduces the process of glycosylation and the impacts of glycans in the aspects of therapeutics. The important glycan structures in therapeutic performances were also summarized focusing on three representative categories of glycoproteins,cytokines, therapeutic antibody and enzyme. Currently, mammalian expression systems such as Chinese hamster ovary cells are preferred for the production of therapeutic glycoproteins due to their ability to synthesize glycans having similar structures with human type glycans. However, recent advances of plant glycoengineering to overcome the limitation originating from different glycan structures will soon allow to develop more efficient and economic plant-based production systems for therapeutic glycoproteins.

Remodeling of the Glycosylation Pathway in the Methylotrophic Yeast Hansenula polymorpha to Produce Human Hybrid-Type N-Glycans

전선아,김현아,오두병,권오석,강현아 한국미생물학회 2012 The journal of microbiology Vol.50 No.2

As a step forward to achieve the generation of human complex-type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc1Man5GlcNAc2 and GlcNAc1Man3GlcNAc2, respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.

Mining and Phylogenetic Analysis of β-1,4-Galactosyltransferase

김성훈,정재갑,오두병,김철호,권오석,강현아 한국생물공학회 2005 한국생물공학회 학술대회 Vol.2005 No.10

Probing the ArcA Regulon in the Rumen Bacterium Mannheimia succiniciproducens by Genome-Wide Expression Profiling

윤슬기,신종문,김오철,정영률,오두병,이상엽,권오석 한국미생물학회 2012 The journal of microbiology Vol.50 No.4

In this study, the putative target genes of the Arc two-component system of the rumen bacterium Mannheimia succiniciproducens were determined by analyzing the transcriptome of the ArcA overexpression strain and by the in silico scanning of the entire genome sequence with the position weight matrix of the ArcA binding sequence developed for Escherichia coli. The majority of 79 repressed genes were involved in energy metabolism and carbohydrate transport and metabolism, while the majority of 82 induced genes were involved in hypothetical or unknown functions. Our results suggest that the Arc system in M. succiniciproducens has a specific function that differs from that in E. coli.

Chimerism of Multiple Monoclonal Antibodies Expressed in a Single Plant

Arshad Jamel,이정환,이경진,오두병,김득수,이경기,추영국,황경아,고기성 한국원예학회 2012 Horticulture, Environment, and Biotechnology Vol.53 No.6

Transgenic plants offer a source for the sustainable, safe, and large-scale production of therapeutic recombinant proteins. In this study, both murine anti-colorectal cancer mAb (mAbMC) and human anti-rabies mAb 57 (mAbHR),expressed in a single plant were investigated for their cancer cell binding activity and rabies virus neutralization activity, respectively. Transgenic plants, expressing murine anti-colorectal cancer mAb CO17-1A (mAbMC) and human anti-rabies mAb 57 (mAbHR), respectively, were crossed to reproduce F1 transgenic plant, expressing both mAbs. PCR and immunoblot analyses demonstrated that heavy (HC) and light chain (LC) genes of mAbMC and mAbHR were present, and that both mAbs were expressed in F1 transgenic lines, respectively. Quantitative immunoblot for purified mAb also showed the presence of both mAbs in F1 transgenic lines. However, Cell ELISA analysis showed that in mAbPC and mAbPR purified from the F1 transgenic lines (mAbPC×R), the binding activity to SW948 human colorectal carcinoma cells was lower than mAbMC, and in vitro mAbMC, mixed with mAbHR (mAbMHC+R). The in vitro rabies virus neutralization assay demonstrated that the mAbPC×R, from the F1 transgenic plants, had lower bioactivity against rabies virus than mAbH57, and mAbMHC+R. N-glycan structure analysis revealed that mAbMC and mAbHR had Golgi type (94 and 14%) and ER type (6 and 86%), respectively, and the purified mAbs from the F1transgenic plants had Golgi type (75%) and ER type (25%). These results indicate that the F1 transgenic plant produced both mAbPC and mAbPR; however, the HC and LC proteins of each anti-rabies virus and anti-colorectal cancer mAbs were assembled randomly, resulting in chimerism in HC and LC assembly for mAb.

Optimization of the human colorectal carcinoma antigen GA733‑2 production in tobacco plants

박세희,지건영,김현민,마상훈,박서영,도주희,오두병,강형식,심재성,정영희 한국식물생명공학회 2021 Plant biotechnology reports Vol.15 No.1

The colorectal carcinoma-associated protein GA733-2 is one of the representative candidate protein for the development of plant-derived colorectal cancer vaccine. Despite of its signifcant importance for colorectal vaccine development, low efciency of GA733-2 production limits its wide applications. To improve productivity of GA733-2 in plants, we here tested multiple factors that afect expression of recombinant GA733-2 (rGA733-2) and rGA733 fused to fragment crystallizable (Fc) domain (rGA733-Fc) protein. The rGA733-2 and rGA733-Fc proteins were highly expressed when the pBINPLUS vector system was used for transient expression in tobacco plants. In addition, the length of interval between rGA733-2 and left border of T-DNA afected the expression of rGA733 protein. Transient expression analysis using various combinations of Agrobacterium tumefaciens strains (C58C1, LBA4404, and GV3101) and tobacco species (Nicotiana tabacum cv. Xanthi nc and Nicotiana benthamiana) revealed that higher accumulation of rGA733-2 and rGA733-Fc proteins were obtained by combination of A. tumefaciens LBA4404 and Nicotiana benthamiana. Transgenic plants generated by introduction of the rGA733-2 and rGA733-Fc expression cassettes also signifcantly accumulated corresponding recombinant proteins. Bioactivity and stability of the plant-derived rGA733 and rGA733-Fc were evaluated by further in vitro assay, western blot and N-glycosylation analysis. Collectively, we here suggest the optimal condition for efcient production of functional rGA733-2 protein in tobacco system.

메탄올 자화효모 Hansenula polymorpha에서의 재조합 단백질 분비발현을 위한 인체 혈청 알부민 융합단편의 활용

송지혜 ( Ji Hye Song ),황동현 ( Dong Hyeon Hwang ),오두병 ( Doo Byoung Oh ),이상기 ( Sang Ki Rhee ),권오석 ( Oh Suk Kwon ) 한국미생물생명공학회(구 한국산업미생물학회) 2013 한국미생물·생명공학회지 Vol.41 No.1

메탄올 자화효모 Hansenula polymorpha에서 분비 발현이 잘 된다고 보고된 인체 혈청 알부민(human serum albumin, HSA)을 융합단편으로 사용하여 외래 재조합 단백질을 효과적으로 분비 발현할 수 있는 발현시스템을 개발하고자 하였다. 이때 조작의 용이성 및 발현 효율 제고를 위하여 전장의 HSA 뿐만 아니라 세 종류의 각기 다른 크기의 HSA 단편을 설계하여 융합단편으로 사용하였다. 즉 HSA의 N-말단으로부터 각기 137, 172, 320, 608개 아미노산을 갖는 융합단편 HSAft (1-4)를 제작하였다. 아울러 발현되는 HSA 단편의 검출 및 분리정제를 위한 His8-tag, HSA 융합단편과 외래 단백질간의 유연성을 부여하기 위한 2조의 Gly4Ser1 linker, 융합 발현된 타겟 단백질을 HSA 단편으로부터 용이하게 분리하기 위한 담배식각바이러스 단백질분해효소(tobacco etch virus protease, Tev) 인지 서열, 타겟 단백질 유전자를 클로닝하기 위한 멀티 클로닝 사이트(multiple cloning site, MCS)서열, 그리고 타겟 재조합 단백질의 발현 검출 및 정제를 위한 Strep-tag을 포함하는 작용기 도메인을 발현카세트 기본 골격에 포함시켰다. 이렇게 구축된 4종의 HSA 융합단편 분비발현 벡터를 H. polymorpha에 형질전환한 후 각 융합단편의 발현을 조사한 결과 HSAft 단편 3, 4의 발현을 확인할 수 있었다. 녹색형광단백질 유전자 (GFPuv)를 상기 벡터에 클로닝한 후 H. polymorpha에 도입한 결과 형질 전환체 모두에서 녹색형광단백질의 발현을 관찰 할 수 있었다. 해당 세포로부터 분비되거나 세포내에 발현되는 HSA 단편융합 형광단백질의 발현양을 비교한 결과 HSAft 단편 4에 융합된 경우를 제외하고 나머지 경우 모두에서 세포 파쇄액과 세포 배양액 양쪽에서 해당 HSA 단편 융합 형광단백질의 발현을 확인 할 수 있었다. 한편 HSA 융합단편의 크기에 따라 자체 혹은 타겟 단백질과 융합된 형태의 단백질 분비 발현 정도가 달라지는 것은 해당 단백질의 접힘이나 단백질 분해효소에 대한 민감성 등 여러 변수에 의한 것으로 사료되며 따라서 본 연구에서 개발한 H. polymorpha용 HSA 융합단편 분비발현 시스템은 특정 외래 재조합 단백질의 효율적인 분비발현 융합단편의 선별 및 과발현 시스템 구축에 유용하게 활용될 수 있을 것으로 기대된다. The thermotolerant methylotrophic yeast Hansenula polymorpha is an attractive model organism for various fundamental studies, such as the genetic control of enzymes involved in methanol metabolism, peroxisome biogenesis, nitrate assimilation, and resistance to heavy metals and oxidative stresses. In addition, H. polymorpha has been highlighted as a promising recombinant protein expression host, especially due to the availability of strong and tightly regulatable promoters. In this study, we investigated the possibility of employing human serum albumin (HSA) as the fusion tag for the secretory expression of heterologous proteins in H. polymorpha. A set of four expression cassettes, which contained the methanol oxidase (MOX) promoter, translational HSA fusion tag, and the terminator of MOX, were constructed. The expression cassettes were also designed to contain sequences for accessory elements including His8-tag, 2×(Gly4Ser1) linkers, tobacco etch virus protease recognition sites (Tev), multi-cloning sites, and strep-tags. To determine the effects of the size of the HSA fusion tag on the secretory expression of the target protein, each cassette contained the HSA gene fragment truncated at a specific position based on its domain structure. By using the Green fluorescence protein gene as the reporter, the properties of each expression cassette were compared in various conditions. Our results suggest that the translational HSA fusion tag is an efficient tool for the secretory expression of recombinant proteins in H. polymorpha.