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Biological Synthesis of Alkyne-terminated Telechelic Recombinant Protein
Ayyadurai, Niraikulam,Kim, So-Yeon,Lee, Sun-Gu,Nagasundarapandian, Soundrarajan,Hasneen, Aleya,Paik, Hyun-Jong,An, Seong-Soo,Oh, Eu-Gene The Polymer Society of Korea 2009 Macromolecular Research Vol.17 No.6
In this study, we demonstrate that the biological unnatural amino acid incorporation method can be utilized in vivo to synthesize an alkyne-terminated telechelic protein, Synthesis of terminally-functionalized polymers such as telechelic polymers is recognized to be important, since they can be employed usefully in many areas of biology and material science, such as drug delivery, colloidal dispersion, surface modification, and formation of polymer network. The introduction of alkyne groups into polymeric material is particularly interesting since the alkyne group can be a linker to combine other materials using click chemistry. To synthesize the telechelic recombinant protein, we attempted to incorporate the L-homopropargylglycine into the recombinant GroES fragment by expressing the recombinant gene encoding Met at the codons for both N- and C-terminals of the protein in the Met auxotrophic E. coli via Hpg supplementation. The Hpg incorporation rate was investigated and the incorporation was confirmed by MALDI-TOF analysis of the telcchelic recombinant protein.
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
Ayyadurai, Niraikulam,Prabhu, Nadarajan Saravanan,Deepankumar, Kanagavel,Jang, Yoon Jung,Chitrapriya, Nataraj,Song, Eunjung,Lee, Nahum,Kim, Seog K.,Kim, Byung-Gee,Soundrarajan, Nagasundarapandian,Lee, American Chemical Society 2011 Bioconjugate chemistry Vol.22 No.4
<P>We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-<SMALL>l</SMALL>-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bcches/2011/bcches.2011.22.issue-4/bc2000066/production/images/medium/bc-2011-000066_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/bc2000066'>ACS Electronic Supporting Info</A></P>
Ayyadurai, Niraikulam,Prabhu, Nadarajan Saravanan,Deepankumar, Kanagavel,Kim, Aran,Lee, Sun-Gu,Yun, Hyungdon Kluwer Academic Publishers 2011 Biotechnology letters. Vol.33 No.11
<P>Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (-8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (-8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.</P>