Most advances in biology can usually be traced back to the development of a new technique: the recent explosion in sequence information in the databases arose from the pioneering work on separation methods by Frederick Sanger which paved the way for the development of protein (Sanger, 1945) and DNA/RNA (Maxam & Gilbert, 1977; Sanger, 1981) sequencing and culminated in the receipt of two Nobel prizes by Sanger. The initial phase of sequence database expansion was slow due to the tedious and slow nature of protein sequencing. Peptide sequencing was carried out manually and the complete analysis of a protein was tiresome, requiring the isolation of sufficient peptides from several digests of the target protein using proteases of different specialities to collect an overlapping set of fragments which cover the whole sequence. Protein sequencing gained momentum when the phenylisothiocyanate sequencing chemistry developed by Edman in 1949 was automated (Edman & Begg, 1967) and a commercial instrument requiring lower amounts (nanomoles) of sample was put on the market. Further technical advances such as novel valves to deal with small volumes of aggressive chemicals, the introduction of high pressure liquid chromatography (HPLC), and novel supports for sample immobilization, were all combined in the first gas phase sequencers, greatly increasing the sensitivity and allowing automated data collection (Hewick et al. 1981) and analysis. The new instruments with a sensitivity in the low picomole range appeared as rapid advances in DNA technology such as the development of restriction mapping (Danna et al. 1973), cloning (Cohen et al. 1973) and the dideoxynucleotide sequencing chemistry were threatening to make protein chemistry a relic of the past (Malcolm, 1978).

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Protein identification in the post-genome era: the rapid rise of proteomics

http://www.jbc.org/content/274/32/22548.full.pdf

Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

/pdf/expression-of-recombinant-human-phenylalanine-hydroxylase-as-pglwnrivn1.pdf

Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme.

Resolution power of two-dimensional electrophoresis and identification of proteins from gels.

Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites.

Sequencing of peptides and proteins from the carboxy terminus

Activation of the carboxy terminus of a peptide for carboxy-terminal sequencing

28 – a comprehensive approach to amino acid analysis

A method of forming a thiohydantoin of formula (II) from an N-protected amino acid (I) is described. The method employs a uronium (III) or phosphonium (IV) compound to activate the terminal carboxyl group of the amino acid and a thiocyanate reagent to cyclize the activated amino acid to the thiohydantoin. The thiohydantoin can be cleaved from its N-protecting group, for use in C-terminal peptide sequencing. Particularly preferred uronium compounds include salts of 2-chlorouronium. Preferred thiocyanate reagents include trimethylsilyl isothiocyanate and crown ether adducts of metallothiocyanates, such as the 18-crown-6 adduct of KSCN.

Method of forming N-protected amino acid thiohydantoins

The alkylated-thiohydantoin method for C-terminal sequencing makes a significant improvement to the thiohydantoin method first described by Schlack and Kumpf Prior to cleavage from the protein, the C-terminal thiohydantoin is alkylated, making it a better leaving group than the unmodified thiohydantoin The C-terminal alkylated-thiohydantoin can be cleaved from the protein under conditions that simultaneously form the next thiohydantoin Combining cleavage and thiohydantoin formation in one step eliminates the need for activating the C-terminal carboxyl group before every sequencing cycle and prevents detection of C-termini formed by random cleavage of peptide bonds in the protein during the sequencing chemistry The alkylated-thiohydantoin method includes the presequencing modification of cysteine and lysine and the automated modification of aspartic and glutamic acids, serine and threonine Modifying the reactive side-chain groups improves the ability to sequence through and detect these amino acids The alkylated-thiohydantoin method can sequence through and detect 19 of the 20 genetically coded amino acids Sequencing stops at proline residues

MeriLisa Bozzini

Papers

Sequencing of peptides and proteins from the carboxy terminus

Activation of the carboxy terminus of a peptide for carboxy-terminal sequencing

28 – a comprehensive approach to amino acid analysis

Method of forming N-protected amino acid thiohydantoins

The alkylated thiohydantoin method for C-terminal sequence analysis.