Purification, nucleotide peptide

Purification of peptides by immobilized enzymes is a relatively recent approach that may have a potential use in the purification of genetically engineered proteins. Purification of recombinant proteins is often difficult and involves many purification steps for a very low yield. Smith et al. found that a specific metal-chelating peptide on the NH2 terminus of a protein can be used to purify that protein with immobilized ion affinity chromatography. The nucleotide sequence that codes for the expressed protein is extended to include codons for the chelating peptide. This concept of amino acid addition to recombinant proteins can also be beneficial in protein purification by affinity chromatography with immobilized enzymes. 

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

Volatile buffers are used for many purposes, particularly in the purification of oligopeptides and oligonucleotides by column chromatography. After the chromatographic separation, the peptides or nucleotides are de-salted and concentrated simply by removing the volatile buffer under vacuum. Table 2-3 lists some commonly used volatile buffers. Because of the concentration which occurs during evaporation of the buffer, it is vital that these buffers are made of very pure components. 

Ion-exchange chromatography has many clinical applications, including the separation of amino acids, peptides, proteins, nucleotides, oligonucleotides, and nucleic acids. Another important application of ion-exchange chromatography is the separation and removal of inorganic ions from aqueous mixtures. Thus most water purification units used to prepare deionized water for the laboratory contain "mixed-bed columns of cation and anion resins (see Chapter 1). 

HPLC life science applications focus on the separation, quantitation, and purification of biomolecules such as proteins, peptides, amino acids, nucleic acids, nucleotides, and polymerase chain reaction (PCR) amplification products.31 34 These are diversified and active research areas in medical research and drug discovery. 

A peptide from pyruvate kinase labeled with oADP (55) and one from ferre-doxin-NADP reductase labeled with oNADP (75) have been isolated and characterized. These are exceptions. Despite the large number of papers describing the kinetics of affinity labeling by periodate-oxidized nucleotides, there are very few reports of the identification of particular amino acids labeled by these reagents within determined peptide sequences. For enzyme products other than a Schiff base reducible by NaBHU, the instability of the products in the proteolytic digests of modified enzymes under conditions of peptide purification has precluded isolation of labeled peptides in most cases. 

As a class of affinity labels, the nucleoside polyphosphopyridoxals exhibit many of the favorable features without many of the drawbacks of the periodate-oxidized nucleotides. The nucleoside polyphosphopyridoxals are structurally close to the natural coenzymes, are water soluble and negatively charged at neutral pH, and exhibit high affinity for the enzymes with which they have been tested. Their targets have thus far been limited to lysine residues, but they react to form Schiff bases which can be stabilized by reduction with NaBH4. Once reduced, the products are stable throughout the peptide purification procedures, and the sequences of the modified peptides can readily be determined. 

As early as 1951, affinity chromatography was used for the separation of anti-hapten antibodies (Campbell et aL, 1951). Shortly afterwards, Lerman (1953) developed a similar method for purification of tyrosinase. The recent applications of affinity chromatography are too numerous to be detailed here, but broadly speaking, the method has been used for separation/purification of proteins, sugars and their derivatives, nucleic acids, nucleotides and their derivatives, amino acids and peptides, and various other systems that include thiols and disulfides (ligands-thiols or disulfides or organomercurial supports), steroidal hormones, coenzymes, vitamins, morphine and related drugs, antibiotics, protein receptors, and antibodies. 

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