Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources

Inteins excise themselves out of precursor proteins by the protein splicing reaction and have emerged as valuable protein engineering tools in numerous and diverse biotechnological applications. Split inteins have recently attracted particular interest because of the opportunities associated with generating a protein from two separate polypeptides and with trans-cleavage applications made possible by split intein mutants. However, natural split inteins are rare and differ greatly in their usefulness with regard to the achievable rates and yields. Here we report the first functional characterization of new split inteins previously identified by bioinformatics from metagenomic sources. The N- and C-terminal fragments of the four inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 were prepared as fusion constructs with model proteins. Upon incubation of complementary pairs, we observed trans-splicing reactions with unprecedented rates and yields for all four inteins. Furthermore, no side reactions were detectable, and the precursor constructs were consumed virtually quantitatively. The rate for the gp41-1 intein, the most active intein on all accounts, was k = 1.8 ± 0.5 × 10(-1) s(-1), which is ～10-fold faster than the rate reported for the Npu DnaE intein and gives rise to completed reactions within 20-30 s. No cross-reactivity in exogenous combinations was observed. Using C1A mutants, all inteins were efficient in the C-terminal cleavage reaction, albeit at lower rates. C-terminal cleavage could be performed under a wide range of reaction conditions and also in the absence of native extein residues flanking the intein. Thus, these inteins hold great potential for splicing and cleavage applications.     

Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Comparing methods for metabolic network analysis and an application to metabolic engineering

Bioinformatics tools have facilitated the reconstruction and analysis of cellular metabolism of various organisms based on information encoded in their genomes. Characterization of cellular metabolism is useful to understand the phenotypic capabilities of these organisms. It has been done quantitatively through the analysis of pathway operations. There are several in silico approaches for analyzing metabolic networks, including structural and stoichiometric analysis, metabolic flux analysis, metabolic control analysis, and several kinetic modeling based analyses. They can serve as a virtual laboratory to give insights into basic principles of cellular functions. This article summarizes the progress and advances in software and algorithm development for metabolic network analysis, along with their applications relevant to cellular physiology, and metabolic engineering with an emphasis on microbial strain optimization. Moreover, it provides a detailed comparative analysis of existing approaches under different categories.