Peptide mapping of HIV polypeptides expressed in Escherichia coli

Renlund, Staffan; Klintrot, Ing-Marie; Nunn, Michael; Schrimsher, Jeffrey L.; Wernstedt, Christer; Hellman, Ulf

doi:10.1016/s0021-9673(01)89499-1

Cited by 14 publications

(4 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The sample (approx. 1.4 mg of purified NADP + -GluDH), dried and re-dissolved in a solution containing 6 M guanidine\HCl, 0.25 M Tris\HCl, pH 8.5, and 1 mM EDTA, was reduced and pyridylethylated [21], and then desalted by HPLC on a 250i4.6 mm Vydac C % column, by using a 0-80 % (v\v) acetonitrile gradient in 0.1 % (v\v) trifluoroacetic acid (TFA), at a flow rate of 1 ml\min.…”

Section: Reduction and Pyridylethylation Of The Nadp + -Gludhmentioning

confidence: 99%

The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression

Barderi

Campetella

Frasch

et al. 1998

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.

show abstract

Section: Reduction and Pyridylethylation Of The Nadp + -Gludhmentioning

confidence: 99%

The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression

Barderi

Campetella

Frasch

et al. 1998

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…The pH for denaturation should maximize protein stability, and the pH for reduction should be optimal for the reducing agent. It is also questionable to have DTT present during denaturation (46,47) as the cysteine bridges are initially not fully accessible and most reduction agents undergo air oxidation (41,48). In addition, oxidized methionine may be fully exposed to the reducing agent for an extensive time.…”

Section: Ph Control During Denaturation and Reductionmentioning

confidence: 99%

“…In 1966 Friedman et al made a study of several such compounds and found that 4VP is an acceptable compromise between reactivity and specificity (82,83). Despite being not as frequently used as IAA, several scientists have pointed out the advantages of 4VP (24,57,84): a complete alkylation with minimal side reactions (85,86) and the possibility to detect cysteine-containing peptides at 254 nm (47). It has been shown that the reactivity of acryl-type reagents with regard to cysteine increases with pH (87).…”

Section: Alkylation With 4-vinylpyridine (4vp)mentioning

confidence: 99%

See 1 more Smart Citation

Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective

Lundell

Schreitmüller

1999

Analytical Biochemistry

135

View full text Add to dashboard Cite

Proteolytic Mapping

Nadler¹

2000

Encyclopedia of Analytical Chemistry

View full text Add to dashboard Cite

Proteolytic mapping is a technique used to verify the primary structure of a protein. It involves the cleavage of the protein at specific amino acids to generate a unique set of peptides which is then separated and analyzed. The chromatogram generated during the separation is known as the proteolytic map of the protein, which serves as a unique “fingerprint” for that protein. A proteolytic map can be used to verify the identity of a protein and to detect mutations in a protein. This technique is faster than two‐dimensional (2D) nuclear magnetic resonance (NMR), X‐ray crystallography, or complete amino acid sequencing for verifying the primary structure of a protein. However, it does not allow determination of the sequence of a protein de novo as can be accomplished with sequencing, nor does it yield the higher order structural information that may be obtained with 2D NMR or X‐ray crystallography. The process of proteolytic mapping involves sample preparation, enzyme digestion, and map development. Each of these steps should be optimized for each new protein to obtain a good map; alternatively, generic protocols can be used to produce satisfactory results for some proteins. A typical protocol requires about a day to complete owing to the time required to digest a protein with an enzyme. However, more recent automated techniques are capable of completing the process in a few hours because they employ a highly concentrated immobilized enzyme which speeds digestion. Proteolytic maps are used in both quality control (QC) and research environments, with the most recent application in the field of proteomics.

show abstract

Peptide mapping of HIV polypeptides expressed in Escherichia coli

Cited by 14 publications

References 10 publications

The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression

The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression

Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective

Proteolytic Mapping

Contact Info

Product

Resources

About