Engineering Protein Sequence Composition for Folding Robustness Renders Efficient Noncanonical Amino acid Incorporations

Nagasundarapandian, Soundrarajan,Merkel, Lars,Budisa, Nediljko,Govindan, Raghunathan,Ayyadurai, Niraikulam,Sriram, Sokalingam,Yun, Hyungdon,Lee, Sun‐,Gu WILEY‐VCH Verlag 2010 Chembiochem Vol.11 No.18

<P><B>Folding up:</B> Residue‐specific incorporation of noncanonical amino acids (NCAAs) often results in loss of protein function either by misfolding or aggregation (left). However, engineering the protein sequence for enhanced folding increases the mutational robustness of the protein to accommodate novel side chains and generate tailor‐made proteins with new properties (right).</P>

Genomewide Analysis of the Antimicrobial Peptides in <i>Python bivittatus</i> and Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity

Kim, Dayeong,Soundrarajan, Nagasundarapandian,Lee, Juyeon,Cho, Hye-sun,Choi, Minkyeung,Cha, Se-Yeoun,Ahn, Byeongyong,Jeon, Hyoim,Le, Minh Thong,Song, Hyuk,Kim, Jin-Hoi,Park, Chankyu American Society for Microbiology 2017 Antimicrobial Agents and Chemotherapy Vol.61 No.9

<P>In this study, we sought to identify novel antimicrobial peptides (AMPs) in Python bivittatus through bioinformatic analyses of publicly available genome information and experimental validation. In our analysis of the python genome, we identified 29 AMP-related candidate sequences. Of these, we selected five cathelicidin-like sequences and subjected them to further in silico analyses. The results showed that these sequences likely have antimicrobial activity. The sequences were named Pb-CATH1 to Pb-CATH5 according to their sequence similarity to previously reported snake cathelicidins. We predicted their molecular structure and then chemically synthesized the mature peptide for three putative cathelicidins and subjected them to biological activity tests. Interestingly, all three peptides showed potent antimicrobial effects against Gram-negative bacteria but very weak activity against Gram-positive bacteria. Remarkably,Delta Pb-CATH4 showed potent activity against antibiotic-resistant clinical isolates and also was observed to possess very low hemolytic activity and cytotoxicity. Delta Pb-CATH4 also showed considerable serum stability. Electron microscopic analysis indicated that. Delta Pb-CATH4 exerts its effects via toroidal pore preformation. Structural comparison of the cathelicidins identified in this study to previously reported ones revealed that these Pb-CATHs are representatives of a new group of reptilian cathelicidins lacking the acidic connecting domain. Furthermore, Pb-CATH4 possesses a completely different mature peptide sequence from those of previously described reptilian cathelicidins. These new AMPs may be candidates for the development of alternatives to or complements of antibiotics to control multidrug-resistant pathogens.</P>

A Comparative Study on the Stability and Structure of Two Different Green Fluorescent Proteins in Organic Co-solvent Systems

Govindan Raghunathan,Sriram Sokalingam,Nagasundarapandian Soundrarajan,Ganapathiraman Munussami,Bharat Madan,이선구 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.2

Green fluorescent protein (GFP) has been used as a reporter marker in a wide range of biological and bioengineering studies. The expanded use of GFP in the field of biosensors, biochips and bio-conjugations requires the stability of GFP in organic co-solvent systems. This prompted us to examine the kinetic stability of two different GFP sequences, n-GFP and s-GFP, showing different folding robustness and thermodynamic stability, under a range of organic co-solvent systems. n-GFP and s-GFP are variants whose biophysical properties are comparable to wild type and super folder GFPs, respectively. The stability of n-GFP and s-GFP in 50% water-miscible organic solvents showed that s-GFP with higher thermodynamic stability exhibited much higher stability against organic solvents than n-GFP, which has lower thermodynamic stability. s-GFP was quite stable even in 90% organic solvents. Circular dichroism analysis confirmed that s-GFP maintained its native structure in organic co-solvent systems, whereas n-GFP showed structural variations under these conditions. Four highly fluctuating loop regions were identified from molecular dynamic simulations under the organic cosolvent conditions. A structural comparison of n-GFP and s-GFP suggested that the improved kinetic stability of s-GFP was due to its larger number of hydrogen bonds and salt-bridges that were present in four loop regions. This study suggests that thermodynamically stable s-GFP can be a good choice for use under harsh organic co-solvent conditions.

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

Modulation of protein stability and aggregation properties by surface charge engineering

Raghunathan, Govindan,Sokalingam, Sriram,Soundrarajan, Nagasundarapandian,Madan, Bharat,Munussami, Ganapathiraman,Lee, Sun-Gu The Royal Society of Chemistry 2013 Molecular bioSystems Vol.9 No.9

<P>An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15–17) and s-GFP(+5–6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.</P> <P>Graphic Abstract</P><P>Engineering surface charge of proteins to modulate the properties of proteins by introducing surface charge mutations with and without stabilizing mutations. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c3mb70068b'> </P>

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.

Synthesis of Well-Defined (Nitrilotriacetic Acid)-End-Functionalized Polystyrenes and Their Bioconjugation with Histidine-Tagged Green Fluorescent Proteins

Cho, Hong Y.,Kadir, Mohammad Abdul,Kim, Bong-Soo,Han, Ho Seok,Nagasundarapandian, Soundrarajan,Kim, Young-Rok,Ko, Sung Bo,Lee, Sun-Gu,Paik, Hyun-jong American Chemical Society 2011 Macromolecules Vol.44 No.12

<P>We have synthesized nitrilotriacetic acid end-functionalized polystyrene (NTA–PS) for the controlled bioconjugation with histidine-tagged green fluorescence proteins (His<SUB>6</SUB>–GFP). NTA–PS was prepared using initiators containing <I>tert</I>-butyl protected NTA moiety via atom transfer radical polymerization (ATRP) of styrene; the protected <I>tert</I>-butyl group was subsequently removed at the α-chain end of polystyrene. The structure of NTA–PS was characterized using <SUP>1</SUP>H NMR, <SUP>13</SUP>C NMR, and GPC. NTA chain ends of the polystyrenes were complexed with Ni<SUP>2+</SUP> to produce Ni–NTA–PS, of which the specific binding properties were studied by forming spherical aggregates with His<SUB>6</SUB>–GFP in aqueous phase. The reversible association of His<SUB>6</SUB>–GFP with polystyrene spherical aggregates (with Ni<SUP>2+</SUP>) was controlled with imidazole and monitored with fluorescence microscope. Again, Ni–NTA–PS produced well-defined micelles with His<SUB>6</SUB>–GFP in water/DMF (DMF 4 vol %) and the size of micelles decreased when excess imidazole was added.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/mamobx/2011/mamobx.2011.44.issue-12/ma200480f/production/images/medium/ma-2011-00480f_0012.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ma200480f'>ACS Electronic Supporting Info</A></P>

Effects of natural resistance-associated macrophage protein 1 and toll-like receptor 2 gene polymorphisms on post-weaning piglet survivability

박찬규,조혜선,김원,최민경,Minh Thong Le,최호준,김진회,김경태,Nagasundarapandian Soundrarajan,박진기,이윤미,김종주 한국유전학회 2016 Genes & Genomics Vol.38 No.2

Predicting resistance or susceptibility to infectious diseases on the basis of the genotypes of animals regarding disease or immune related genes could be important in animal production. We evaluated the potential influence of the genetic polymorphisms of four immune related genes, porcine beta defensin 4, interferon-induced GTP-binding protein Mx1, natural resistance-associated macrophage protein 1 (Nramp1), and Toll-like receptor 2 (TLR2) on post-weaning piglet survivability in farms. We selected a single SNP that satisfied the criteria of nonsynonymous substitutions and the minor allele frequency [0.1 from each gene, and performed PCR–RFLP. Distributions of allele and genotype frequencies of the SNPs were, in general, significantly different among the five breeds (Berkshire, Yorkshire, Duroc, Landrace, and Korean native pigs. Initially, 371 randomly collected Yorkshire 9 Landrace F1 piglets consisting of post-weaning survival (n = 185) and non-survival groups (n = 186) were genotyped for the selected SNPs. For the Nramp1 and TLR2 SNPs, genotype frequencies were significantly different between the two groups (P\0.05). To confirm the results, additional animals that were collected in a different time-period were genotyped for the Nramp1 (n = 390) and TLR2 (n = 240) SNPs. The results using the combined samples (n = 761) were consistent with the initial analysis (P\0.01), suggesting that the genetic polymorphisms of Nramp1 and TLR2 affect post-weaning piglet survivability. The relative risks, i.e. odds ratios, between the beneficial and non-beneficial genotypes to piglet survivability were 4.88 and 29.65 for the Nramp1 and TLR2 SNPs, respectively.

Bioconjugation of <small>l</small>-3,4-Dihydroxyphenylalanine Containing Protein with a Polysaccharide

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>