The S30 extract from E. coli BL21 Star (DE3) used for cell-free protein synthesis removes a wide range of α-amino acid protecting groups by cleaving α-carboxyl hydrazides; methyl, benzyl, tert-butyl, and adamantyl esters; tert-butyl and adamantyl carboxamides; α-amino form-, acet-, trifluoroacet-, and benzamides; and side-chain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)-4-fluoroleucine, (S)-4,5-dehydroleucine, and (2S,3R)-4-chlorovaline into a protein through the direct use of their respective precursors, namely, (S)-4-fluoroleucine hydrazide, (S)-4,5-dehydroleucine hydrazide, and (2S,3R)-4-chlorovaline methyl ester. These results also show that the fluoro- and dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively.

Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.

Native and engineered protein biosynthetic machinery processes a wide range of fluorinated a-amino acids for incorporation into peptides and proteins, either as substitutes for structurally similar amino acids normally found in proteins, or as additional ones. In the former case, replacement occurs wherever the normal amino acid is encoded, while the latter method is site-specific. The fluorinated peptides have a diverse variety of interesting properties. The biochemical synthetic methods are straightforward, to the point that they should routinely be assessed as alternatives to traditional solid-and solution-phase peptide synthesis.

Hydraulic fracture stimulation of low permeability source rock-reservoirs is increasing in the industry and low-permeability, deeply buried coals, "Deep Coals," represent an underdeveloped resource in the Cooper Basin of South Australia. Numerous treatments have been performed with overall technical success but varied productivity. Thus, it was important to understand the potential hydraulic fracture conductivity in the Permian Deep Coal play by evaluating the key treatment variables affecting post-stimulation results. Proppant conductivity tests were conducted on samples from two preserved Deep Coal cores of differing thermal maturity under laboratory conditions, replicating reservoir conditions. Varying concentrations and mesh sizes of lightweight ceramic (LWP) and sand proppants were tested at 250°F. All tests were subject to closure stresses observed in the field, ranging from 2,000 psi to 10,000 psi, using representative hydraulic fracturing fluids. Results revealed a significant conductivity difference between the two coal thermal ranks due to variation in mechanical properties Varying proppant concentration tests revealed that effective conductivity at higher stresses occurs within a narrow window. This window is a balance between insufficiently low concentration resulting in significant conductivity loss, and excessive, non-linear conductivity gains in higher concentrations. Results from these studies are integrated into a hydraulic fracture and reservoir stimulation modelling software to up-scale the observed results versus the laboratory data. Finally, laboratory results helped explain trends for success in stimulation design based on observed post-frac, flow rates.

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