Sequencing, proteins recovery

Proteins produced in plant cells can remain within the cell or are secreted into the apoplast via the bulk transport (secretory) pathway. In whole plants, because levels of protein accumulated intracellularly, e. g. using the KDEL sequence to ensure retention in the endoplasmic reticulum, are often higher than when the product is secreted [58], foreign proteins are generally not directed for secretion. However, as protein purification from plant biomass is potentially much more difficult and expensive than protein recovery from culture medium, protein secretion is considered an advantage in tissue culture systems. For economic harvesting from the medium, the protein should be stable once secreted and should accumulate to high levels in the extracellular environment. 

Another example of separating long Tg components from short Tg components is given by Rabenstein, et al. (1979). They named the sequence inversion-recovery spin-echo (IRSE) because it is literally just that. Instead of the n/2 pulse to monitor the magnetization recovering after the initial 7t pulse, a n-n/2 echo is used. The separation of the two pulses in the spin echo sub-sequence is adjusted so that the short T2 component does not contribute significantly to the echo. This sequence was successfully used to isolate the sharp NMR lines of small molecules from the broad lines of proteins in solutions. 

Alterations to the proteins and pre-proteins expressed by cultured plant cells have been used to facilitate product recovery. A leader sequence is required for foreign protein secretion from plant cells into the apoplast and then into the culture medium. As indicated in Table 2.1, plant, mammalian and viral sequences have been employed to achieve the entry of transgenic proteins into the bulk-flow pathway in plant cultures. 

Fig. 14. SeqHNCA-TROSY experiment for establishing sequential 1HN(i), 15N(i), 13Ca(i— 1) correlations in 13C/15N/2H enriched proteins. Durations of transfer delays A = 1/(4/Hn) 2Ta = 20-27 ms, depending on rotational correlation time of protein 2Tc = 5-7 ms S = gradient + field recovery delay 0 < k < Ta/t2,max- Phase cycling i = y (j>2 = y, — y + States-TPPI 0 = x 0ret. = x, — x. Semi-selective decoupling of 13C spins is attained using a SEDUCE-1 decoupling sequence.95...

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

Ribonuclease A (RNase A) was selected as the target enzyme for solid-phase synthesis because its sequence was known (Scheme S), 22 25 and an X-ray structure had been deduced. 24 Importantly, it had been shown that this 124-residue protein could be reduced and unfolded and then reoxidized to re-form the four disulfide bonds with recovery of full enzymatic activity. 25 ... 

Certain polypeptides resulting from protease digestion of proteins contribute to the typical taste of savoury foods. The DNA sequence coding for an octapep-tide known as beefy meaty peptide was cloned into yeast as a fusion with the yeast a factor to be secreted as free octapeptide into the medium which facilitated its recovery [37]. Alternatively, intracellular expression of tasty peptides... 

An alternative to the tank transfer system is the semidry transfer system. In this procedure, the gel is stacked horizontally on top of the membrane in the transfer apparatus. Because only a small volume of transfer buffer is used, SDS from the gel is less effectively diluted, which may result in incomplete binding and lower yields, especially with PVDF membranes. For this reason, semidry transfer units are not recommended when reproducible high recoveries of electroblotted proteins are desired (e.g., for subsequent sequence analysis). Some procedures recommend stacking multiple transfer sandwiches to achieve several transfers simultaneously. To prevent unbound protein from migrating through the next gel and onto the membrane in the next transfer stack, sheets of porous cellophane sheets or dialysis membrane are placed between adjacent transfer stacks (see Fig. B3.2.3). Semidry electrotransfer requires shorter transfer times than tank transfer. 

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