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The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein's stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89-93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His 6 fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.

Proteins are an increasingly important class of new drugs. Pharmaceutical proteins are usually expressed in cell based systems in the development phase and in production, and although cell free methods have recently emerged they have not been used widely for therapeutic protein development or production. Cell free expression methodology is well suited for pharmaceutical protein expression and engineering and will probably become more commonly used in the future. Cell free expression allows protein engineering in high throughput format, flexible strategies for glycosylation and chemical conjugation, and allows easy use of unnatural amino acids as building blocks of proteins. Thus, cell free expression can be used to modify protein solubility, stability, and pharmacokinetics of therapeutic proteins. Likewise, it is potentially useful in protein development for biomaterial matrices, nanoparticles, and vaccines. This review illustrates the potential of cell free expression in pharmaceutical protein research and development while highlighting both advantages and limitations of the method., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove.