From the notebook to recombinant protein production in Escherichia coli: Design of expression vectors and gene cloning.

Research in recombinant protein expression in microorganism hosts spans half a century. The field has evolved from mostly trial-and-error approaches to more rational strategies, including careful design of the expression vectors and the coding sequence for the protein of interest. It is important to reflect on many aspects about vector construction, such as codon usage, integration site, coding sequence mutagenesis and many others. In this chapter, we overview methods and considerations to generate a suitable construct and anticipate possible experimental roadblocks.

Starting a new recombinant protein production project in Escherichia coli.

One of the goals in recombinant protein production in Escherichia coli is to maximize productivity. High volumetric and specific yields can be reached after careful selection of expression strains and optimization of cultivation parameters. In this chapter, we review the many tools available to make the most out of this versatile microbial cell factory. Useful guidelines and options for troubleshooting production are presented.

Structural features of the plant N-recognin ClpS1 and sequence determinants in its targets that govern substrate selection.

In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway.