The Use of Carbohydrate Protein Conjugates of Proteases [CPC(Proteases)] for the Catalytic Formation of Peptide Bonds

1990 ◽  
Vol 218 ◽  
Author(s):  
Peng Wang ◽  
Tara G. Hill ◽  
Mark D. Bednarski ◽  
Matthew R. Callstrom
Keyword(s):  
Proteolytic Enzymes ◽  
Peptide Bond ◽  
Peptide Sequence ◽  
Covalent Attachment ◽  
General Strategy ◽  
Protein Chemistry ◽  
Peptide Fragments ◽  
Peptide Bonds ◽  
Protein Conjugates ◽  
The Stability

The discovery of catalysts that can selectively couple unprotected peptide fragments would revolutionize protein chemistry by allowing convergent polypeptide synthesis. Proteolytic enzymes have the capability to perform this chemistry because the protein can specifically recognize and bind to C-terminal and N-terminal peptide sequences, activate the C-terminal peptide sequence by forming an acyl-enzyme intermediate, and couple the two peptide fragments together. However, barriers that limit the use of proteases as catalysts for convergent peptide synthesis include (i) the stability of proteolytic enzymes in organic solvent systems; (ii) a simple and effective C-erminal and N-terminal protecting group strategy; and (iii) the isolation of the polypeptide product from the reaction mixture. In the previous paper we reported the stabilization of enzymes by the covalent attachment of proteins through their ο-lysine residues to a series of carbohydrate-based macromolecules. In this paper we report the use of carbohydrate protein conjugates of proteases [CPC(proteases)] as catalysts for peptide bond synthesis and a general strategy for convergent oligopeptide synthesis.

scholarly journals Effect of Mutations in the Human Immunoglobulin A1 (IgA1) Hinge on Its Susceptibility to Cleavage by Diverse Bacterial IgA1 Proteases

2005 ◽  
Vol 73 (3) ◽  
pp. 1515-1522 ◽  
Author(s):  
Bernard W. Senior ◽  
Jenny M. Woof
Keyword(s):  
Peptide Bond ◽  
Human Immunoglobulin ◽  
Peptide Sequence ◽  
Wild Type ◽  
Peptide Bonds ◽  
Streptococcus Sanguis ◽  
In The Wild ◽  
Scissile Bond ◽  
Cleavable Peptide

ABSTRACT Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases.

Synthesis of the carboxy-terminal octapeptide of cholecystokinin (CKK-8) based on incorporation of O4-sulfotyrosine by enzymatically catalyzed formation of peptide bonds

1988 ◽  
Vol 53 (5) ◽  
pp. 1086-1093 ◽  
Author(s):  
Václav Čeřovský ◽  
Jan Hlaváček ◽  
Jiřina Slaninová ◽  
Karel Jošt
Keyword(s):  
Aspartic Acid ◽  
Sodium Salt ◽  
Proteolytic Enzymes ◽  
Biological Activities ◽  
Proteinase K ◽  
Side Chain ◽  
Peptide Fragments ◽  
Peptide Bonds ◽  
Carboxy Terminal ◽  
Amide Fragment

Papain-catalyzed condensation of sodium salt of tert-butyloxycarbonyl-β-tert-butyloxyaspartyl-O4-sulfotyrosine (fragment 1-2) with methionyl-glycyl-tryptophyl-methionyl-aspartyl-phenylalanine amide (fragment 3-8) has been elaborated. Deprotection of the thus-obtained octapeptide afforded CCK-8 which exhibited full biological activities. Benzyloxycarbonylaspartyl-phenylalanine amide (fragment 7-8) was prepared using thermolysin without protecting the aspartic acid side chain. Attempted condensation of tert-butyloxycarbonylmethionyl-glycyl-tryptophan (fragment 3-5) with methionyl-aspartyl-phenylalanine amide (fragment 6-8), catalyzed by α-chymotrypsin, subtilisin or proteinase K, afforded the product (fragment 3-8) in only low yields. Further use of proteolytic enzymes for preparing other peptide fragments of the CCK-8 molecule without side chain protection is investigated.

scholarly journals Redirecting RiPP biosynthetic enzymes to proteins and backbone-modified substrates

2022 ◽  
Author(s):  
Joshua A Walker ◽  
Noah Hamlish ◽  
Avery Tytla ◽  
Daniel D Brauer ◽  
Matthew B Francis ◽  
...  
Keyword(s):  
Natural Products ◽  
Peptide Bond ◽  
Chemical Diversity ◽  
Conformational Space ◽  
General Strategy ◽  
Anticancer Properties ◽  
Modified Peptides ◽  
The Stability ◽  
Polypeptide Chains ◽  
Enzyme Class

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products that include the FDA-approved analgesic ziconotide1,2 as well as compounds with potent antibiotic, antiviral, and anticancer properties.3 RiPP enzymes known as cyclodehydratases and dehydrogenases represent an exceptionally well-studied enzyme class.3 These enzymes work together to catalyze intramolecular, interresidue condensation3,4 and aromatization reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomers, including aramids and beta-amino acids, within leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse, non-alpha-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bond. Both features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. Moreover, as thiazoles and thiazoline heterocycles are replete in natural products,5,6,7 small molecule drugs,8,9 and peptide-mimetic therapeutics,10 their installation in protein-based biotherapeutics could improve or augment performance, activity, stability, and/or selectivity. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

672 An Investigation of Antibiotic Loading and Release in Acid-precipitated Keratin Proteins to Treat Topical Burn Infections

2021 ◽  
Vol 42 (Supplement_1) ◽  
pp. S192-S193
Author(s):  
Marc A Thompson ◽  
Robert J Christy
Keyword(s):  
Proteolytic Enzymes ◽  
Diglycidyl Ether ◽  
Water Soluble ◽  
Bacterial Colony ◽  
Freeze Dried ◽  
Antibiotic Compounds ◽  
Water Ph ◽  
Stable Product ◽  
Antibiotic Release ◽  
The Stability

Abstract Introduction Human-hair derived keratin (KOS) protein has been selected in this investigation for its ability to bind antibiotic compounds and provide sustained release while withstanding harsh proteolytic environments such as inflamed, damaged tissue. The need to control local flora has been recognized as an imperative for wound healing, as recovery is significantly hampered by infection. This study investigates the synthesis of KOS-based particulate matter, developed using acid-precipitation, to load and release the water-soluble antibiotic ciprofloxacin (CIP). We hypothesize that ionically bound CIP release is tied to the degradation of KOS, therefore, bacterial metabolism, which produces proteolytic enzymes, will trigger CIP release thereby creating a novel self-extinguishing delivery system for contaminated skin wounds. Methods Ciprofloxacin hydrochloride was solubilized in deionized water (pH 5.3) under constant stirring. Freeze-dried KOS powder was added for an ultimately 5% w/v and 0.8% w/v solution of KOS and CIP, respectively. To improve the stability of KOS a water-soluble diglycidyl ether crosslinker was added to solutions and stirred for 24 hours. CIP-loaded protein was precipitated out by a hydrochloric acid induced pH reduction. Samples were collected and frozen at -20 °C prior to lyophilization, thus forming the stable product. Degradation of KOS and commensurate release of CIP were measured using a bicinchoninic acid (BCA) assay and fluorescent measurements of hydrated material supernatant. The reduction of bacterial colonies was validated by a broth inhibition assay whereby CIP-loaded KOS or unloaded KOS controls where hydrated in bacterial-laden broth cultures of Pseudomonas aeruginosa or Methicillin-resistant Staphylococcus aureus. Cultures were sampled at 24, 48, or 72 hours and plated to quantify colony-forming units. Results The presence of CIP in the KOS protein was confirmed and release rates follow similar patterns to that of KOS degradation. CIP-loaded proteins significantly reduce bacterial colony presence in concentrated inoculant solutions up to 72 hours. Conclusions CIP release does appear to coincide with KOS degradation, which is bolstered in the presence of infectious levels of bacteria. Ongoing studies aim to observe more robust models of infection and more controlled antibiotic release.

scholarly journals Isolation, purification and structure of exochelin MS, the extracellular siderophore from Mycobacterium smegmatis

1995 ◽  
Vol 305 (1) ◽  
pp. 187-196 ◽  
Author(s):  
G J Sharman ◽  
D H Williams ◽  
D F Ewing ◽  
C Ratledge
Keyword(s):  
Amino Acids ◽  
Mycobacterium Smegmatis ◽  
Methyl Esters ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Gallium Complex

The extracellular siderophore from Mycobacterium smegmatis, exochelin MS, was isolated from iron-deficiently grown cultures and purified to > 98% by a combination of ion-exchange chromatography and h.p.l.c. The material is unextractable into organic solvents, is basic (pI = 9.3-9.5), has a lambda max at 420 nm and a probable Ks for Fe3+ of between 10(25) and 10(30). Its structure has been determined by examination of desferri- and ferri-exochelin and its gallium complex. The methods used were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-COSY and TOCSY) 1H n.m.r. The constituent amino acids were examined by chiral g.l.c analysis of N-trifluoroacetyl isopropyl and N-pentafluoropropionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(delta-N-formyl,delta N-hydroxy-R-ornithyl) -beta-alaninyl-delta N-hydroxy-R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linkages involving the three ornithine residues are via their delta N(OH) and alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thus the molecule has no conventional peptide bond, and this suggests that it will be resistant to peptidase hydrolysis. The co-ordination centre with Fe3+ is hexadenate in an octahedral structure involving the three hydroxamic acid groups. Molecular modelling shows it to have similar features to other ferric trihydroxamate siderophores whose three-dimensional structures have been established. The molecule is shown to have little flexibility around the iron chelation centre, although the terminal (Orn-3) residue, which is not involved in iron binding except at its delta N atom, has more motional freedom.

scholarly journals The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

2021 ◽  
Vol 12 ◽  
Author(s):  
Pia Gattinger ◽  
Shiva Izadi ◽  
Clemens Grünwald-Gruber ◽  
Somanath Kallolimath ◽  
Alexandra Castilho
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Degradation Products ◽  
Therapeutic Proteins ◽  
Cost Effective ◽  
Full Length ◽  
Peptide Sequence ◽  
Reporter Protein ◽  
The Stability

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

Isotope-Edited Raman Spectroscopy of Proteins:  A General Strategy To Probe Individual Peptide Bonds with Application to Insulin

2001 ◽  
Vol 123 (32) ◽  
pp. 7919-7920 ◽  
Author(s):  
Jian Dong ◽  
Zhu-Li Wan ◽  
Ying-Chi Chu ◽  
Satoe N. Nakagawa ◽  
Panayotis G. Katsoyannis ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
General Strategy ◽  
Peptide Bonds ◽  
Individual Peptide

scholarly journals The complete amino acid sequence of chicken skeletal-muscle enolase

1986 ◽  
Vol 236 (1) ◽  
pp. 115-126 ◽  
Author(s):  
G A Russell ◽  
B Dunbar ◽  
L A Fothergill-Gilmore
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Peptide Bond ◽  
Peptide Bonds ◽  
Complete Amino Acid Sequence ◽  
Arginine Residues ◽  
Cnbr Cleavage ◽  
Yeast Enolase ◽  
Chicken Skeletal Muscle

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].

scholarly journals The Reactivity and Allergenic Potential of Hazelnut Peptides

2013 ◽  
Vol 70 (1) ◽  
pp. 25
Author(s):  
Lavinia Florina Calinoiu ◽  
Dan Cristian Vodnar ◽  
Carmen Socaciu
Keyword(s):  
Proteolytic Enzymes ◽  
Peptide Fragments ◽  
Dot Blot ◽  
Specific Antibodies ◽  
Allergenic Potential ◽  
Sds Page ◽  
Allergenic Proteins ◽  
Life Threatening ◽  
Specific Proteins ◽  
Sensitive Method

The aim of this paper was to focus on proteins present in some food products, like hazelnuts and to investigate their allergenic potential. Several techniques were used to characterize these extracted proteins, with respect to their composition, degradability by digestive proteolytic enzymes and their reactivity with specific antibodies. It was important to analyse which proteins were present in the hazelnuts, to see if there were proteins present to trigger an allergic reaction and if the digestion enzymes trypsin and pepsin influence the presence of the (allergic) protein compounds. Allergies to tree nuts and seeds can cause life-threatening and sometimes fatal reactions. To examine the properties of Hazelnut protein it was important to solubilize it by extraction. After extraction, it was investigated how hazelnut protein can be modified by proteases and what the effect was on the immune reaction. The Bradford method is a fast and sensitive method to determine the concentration of soluble protein. When the Bradford reagent (Coomassie Brilliant Blue) binds to the protein, the colour changes from red to purple and the absorption maximum changes from 495 to 595 nm. The value obtained as the final concentration of proteins was 7.3495. SDS-PAGE is a method to separate mixtures of proteins by electrophoresis. Protein molecules are negatively charged by binding of SDS molecules; subsequently they are separated in an electric field. Their differences in size (molecular weight) leads to separation. In this case the method is used to follow proteolytic degradation of hazelnut proteins (allergens) by intestinal proteases (trypsin, pepsin). A different, more specific and sensitive method is immunoblotting (Western Blot) in which the SDS-PAGE separated proteins are transferred from the gel to a membrane and specific antibodies are used in a series of reactions to visualize specific allergens on this membrane. The remarked spots represented a positive identification of allergenic proteins. This means that peptide fragments of various size, produced during the digestion of a protein can still be immunological active. As it was shown there was still reactivity between proteins and specific antibodies. The Dot Blot is a simple immunoblotting technique used to detected specific proteins in a mixture of different proteins and/or other molecules. No separation technique prior to the actual immuno-detection is necessary. Also, Dot Blot confirmed the presence of allergenic proteins made visible through the light spots on the membrane.

Sign in / Sign up

Export Citation Format

Share Document