NP-Sticky: a web server for optimizing DNA ligation with non-palindromic sticky ends.

High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize specificity. However, optimizing such multi-parametric ligation reactions often involves extensive trial and error. We have developed a freely available Web-based ligation calculator, NP-Sticky (http://sarkarlab.umn.edu/npsticky/), that predicts product distribution for given reactant concentrations, thus enabling straightforward computational optimization of these reactions. Built-in schemes include two-piece and three-piece linear ligation, as well as insert-vector circular ligation. The only parameters needed for the underlying thermodynamic model are the free energies of ligation for each sticky end, which can be estimated by the calculator from the overhang sequences or provided by the user from direct experimental measurement. Free energies of sticky-end mismatches are also calculated for determining the extent of byproduct formation. This ligation calculator allows rapid identification of the optimal conditions for maximizing incorporation, efficiency, and/or accuracy, based on specific needs.

Streamlined protocol for mRNA display.

mRNA display is a powerful method for in vitro directed evolution of polypeptides, but its time-consuming, technically demanding nature has hindered its widespread use. We present a streamlined protocol in which lengthy mRNA purification steps are replaced with faster precipitation and ultrafiltration alternatives; additionally, other purification steps are entirely eliminated by using a reconstituted translation system and by performing reverse transcription after selection, which also protects input polypeptides from thermal denaturation. We tested this procedure by performing affinity selection against Her2 using binary libraries containing a nonspecific designed ankyrin repeat protein (DARPin) doped with a Her2-binding DARPin (dopant fraction ranging from 1:10 to 1:10 000). The Her2-binding DARPin was recovered in all cases, with an enrichment factor of up to 2 orders of magnitude per selection round. The time required for 1 round is reduced from ∼4-7 days to 2 days with our protocol, thus simplifying and accelerating mRNA display experiments.

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

Effective phagocytosis of low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcγRIIa affinity and selectivity.

Glycans anchored to residue N297 of the antibody IgG Fc domain are critical in mediating binding toward FcγRs to direct both adaptive and innate immune responses. However, using a full length bacterial IgG display system, we have isolated aglycosylated Fc domains with mutations that confer up to a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous human inhibitory receptor, FcγRIIb. The mutant Fc domain (AglycoT-Fc1004) contained a total of 5 amino acid substitutions that conferred an activating to inhibitory ratio of 25 (A/I ratio; FcyRIIa-R131:FcγRIIb). Incorporation of this engineered Fc into trastuzumab, an anti-Her2 antibody, resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with both medium and low Her2-expressing cancer cells. A mathematical model has been developed to help explain how receptor affinity and the A/I ratio relate to improved antibody dependent cell-mediated phagocytosis. Our model provides guidelines for the future engineering of Fc domains with enhanced effector function.

Model-guided ligation strategy for optimal assembly of DNA libraries.

DNA ligation is essential to many molecular biology manipulations, but this reaction is often carried out by following generic guidelines or by trial and error. Maximizing the desired ligation product is especially important in DNA library construction for directed evolution experiments since library diversity is directly affected by ligation efficiency. Here, we suggest that display vectors that rely on Type IIP restriction sites for cloning should be redesigned to utilize Type IIS restriction sites instead because ligation yield is significantly improved: we observed up to 15- and 2.6-fold increases in desired products for circular and linear ligation reactions, respectively. To guide ligation optimization more rationally, we developed an easily parameterized thermodynamic model that predicts product distributions based on input DNA concentrations and free energies of the ligation events. We applied this model to study ligation reactions using a ribosome display vector redesigned with Type IIS restriction sites (pRDV2). We computationally predicted and experimentally validated the relative abundance of various products in three-piece linear ligations as well as the extent of transformation from vector-insert circular ligations. Based on our results, we provide general insights into ligation and we outline guidelines for optimizing this reaction for both in vivo and in vitro display methodologies.

Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000.

Oral delivery of insulin to diabetic patients is highly desirable because it would be non-invasive and more closely mimic normal physiology, but this route of administration typically results in low bioavailability due to low pH and enzymatic degradation along the gastrointestinal tract. To explore an alternative approach that may mitigate these obstacles and also facilitate local synthesis of new therapeutic protein molecules in the small intestine, we engineered the food-grade bacterium Lactococcus lactis (NZ9000) for nisin-inducible expression and secretion of a bioactive single-chain insulin (SCI) analog, SCI-57. We show that the addition of nisin during early-log phase has a modest inhibitory effect on cell growth but induction during mid-log phase has a negligible impact on proliferation, suggesting a tradeoff between cell growth rate and duration of induction. We find that a signal peptide such as usp45 is necessary for secretion of SCI-57 into the medium; furthermore, we demonstrate that this secreted SCI-57 is biologically active, as assessed by the ability of conditioned L. lactis medium to stimulate Akt signaling in differentiated 3T3-L1 adipocytes. Finally, we show that the biological activity of SCI-57 was enhanced by near-neutral or slightly alkaline pH during induction, which is comparable to the pH in the small intestine, and by removal of a C-terminal purification tag. This study demonstrates that food-grade bacteria can be engineered to secrete bioactive insulin analogs and opens up the possibility of oral insulin delivery using live microorganisms.

Effects of overexpression of X-box binding protein 1 on recombinant protein production in Chinese hamster ovary and NS0 myeloma cells.

X-box binding protein 1 (XBP-1) is a key regulator of the cellular secretory pathway and unfolded protein response (UPR). It has been shown that the spliced form of XBP-1, XBP-1S, functions as a transcription activator and up-regulates many genes associated with protein secretion and biosynthesis of endoplasmic reticula. Since the production of some recombinant proteins is widely believed to be limited by the secretory capacity of the host cell, an increase in protein production may be achieved by overexpressing XBP-1S. In this study, the effects of XBP-1S on the productivity of monoclonal antibody (MAb), interferon gamma (IFNgamma), and erythropoietin (EPO) are examined in Chinese hamster ovary (CHO) and NS0 cell lines. Results show that XBP-1S may become a determinative factor only when accumulation of recombinant proteins exceeds the secretory capacity of the host cell. In transient transfection systems where a bottleneck in protein secretion was achieved, overexpression of XBP-1S improved protein titers by up to 2.5-fold. In contrast, overexpression of XBP-1S had no detectable effects on protein productivity of stable cell lines that did not exhibit any secretory bottleneck. We conclude that overexpression of XBP-1S is an effective strategy in enhancing recombinant protein production when the secretory pathway of the host cell is saturated by high-level synthesis of recombinant proteins.