Protein Engineering Design and Selection - current issue

Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs

2008-04-14

We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa ‘AlbudAbs’ can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.

Genetic selection for peptide inhibitors of angiogenin

Smith, B. D., Raines, R. T. — 2008-04-14

The improper regulation of angiogenesis is implicit in a variety of diseases, including cancer. Angiogenin is unique among angiogenic factors in having ribonucleolytic activity. Inhibitors of this activity could serve as chemotherapeutics. The ribonucleolytic activity of angiogenin is toxic to the Origami^TM strain of Escherichia coli. Herein, this cytotoxicity was used to identify inhibitors from a random nonapeptide library tethered to the C-terminus of human angiogenin. The selected sequences fell into three classes: (i) extremely hydrophobic, (ii) putative protease (ClpXP) substrates and (iii) slightly anionic. Two peptides corresponding to sequences in the last class were synthesized chemically and found to inhibit the ribonucleolytic activity of human angiogenin in vitro with micromolar values of K_i. Both peptides also inhibit bovine pancreatic ribonuclease, a homolog of angiogenin, though one exhibits selectivity for angiogenin. The affinity and selectivity of these peptides are comparable with the best known inhibitors of angiogenin. Moreover, the strategy used to identify them is general and could be applied to other cytotoxins.

An improved prediction of catalytic residues in enzyme structures

Tang, Y.-R., Sheng, Z.-Y., Chen, Y.-Z., Zhang, Z. — 2008-04-14

The protein databases contain a huge number of function unknown proteins, including many proteins with newly determined 3D structures resulted from the Structural Genomics Projects. To accelerate experiment-based assignment of function, de novo prediction of protein functional sites, like active sites in enzymes, becomes increasingly important. Here, we attempted to improve the prediction of catalytic residues in enzyme structures by seeking and refining different encodings (i.e. residue properties) as well as employing new machine learning algorithms. In particular, considering that catalytic residues can often reveal specific network centrality when representing enzyme structure as a residue contact network, the corresponding measurement (i.e. closeness centrality) was used as one of the most important encodings in our new predictor. Meanwhile, a genetic algorithm integrated neural network (GANN) was also employed. Thanks to the above strategies, our GANN predictor demonstrated a high accuracy of 91.2% in the prediction of catalytic residues based on balanced datasets (i.e. the 1:1 ratio of catalytic to non-catalytic residues). When the GANN method was optimally applied to real enzyme structures, 73.9% of the tested structures had the active site correctly located. Compared with two existing methods, the proposed GANN method also demonstrated a better performance.

Thermostability promotes the cooperative function of split adenylate kinases

Nguyen, P. Q., Liu, S., Thompson, J. C., Silberg, J. J. — 2008-04-14

Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AK_Bs) and the hyperthermophile Thermotoga neapolitana (AK_Tn). Complementation studies revealed that the split AK_Tn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AK_Bs. However, weak complementation occurred when the AK_Bs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AK_Tn had higher levels of zinc, greater secondary structure, and >10³-fold more activity than the AK_Bs pair, albeit 17-fold less active than full-length AK_Tn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

Amino acid alphabet size in protein evolution experiments: better to search a small library thoroughly or a large library sparsely?

Munoz, E., Deem, M. W. — 2008-04-14

We compare the results obtained from searching a smaller library thoroughly versus searching a more diverse, larger library sparsely. We study protein evolution with reduced amino acid alphabets, by simulating directed evolution experiments at three different alphabet sizes: 20, 5 and 2. We employ a physical model for evolution, the generalized NK model, that has proved successful in modeling protein evolution, antibody evolution and T-cell selection. We find that antibodies with higher affinity are found by searching a library with a larger alphabet sparsely than by searching a smaller library thoroughly, even with well-designed reduced libraries. We also find ranked amino acid usage frequencies in agreement with observations of the CDR-H3 variable region of human antibodies.

Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase

2008-04-14

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis–Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher k_cat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the T_m values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E.

Diverging catalytic capacities and selectivity profiles with haloalkane substrates of chimeric alpha class glutathione transferases

Kurtovic, S., Shokeer, A., Mannervik, B. — 2008-04-14

Six homologous Alpha class glutathione transferases of human, bovine, and rat origins were hybridized by means of DNA shuffling. The chimeric mutants were compared with the parental enzymes in their activities with several alkyl iodides. In order to facilitate a multivariate analysis of relationships between substrates and enzyme activities, three descriptors were introduced: ‘specific catalytic capacity’, ‘substrate selectivity’, and ‘unit-scaled substrate selectivity’. In some cases the purified mutants showed higher specific activity with a certain alkyl iodide than any of the parental enzymes. However, the overriding effect of DNA shuffling was the generation of chimeras with altered substrate selectivity profiles and catalytic capacities. The altered substrate selectivity profiles of some mutants could be rationalized by changes of the substrate-binding residues in the active site of the enzyme. However, in four of the isolated mutants all active-site residues were found identical with those of rat GST A2-2, even though their substrate specificity profiles were significantly different. Clearly, amino acid residues distant from first-sphere interactions with the substrate influence the catalytic activity. These results are relevant both to the understanding how functional properties may develop in natural enzyme evolution and in the tailoring of novel functions in protein engineering.

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants

Chodorge, M., Fourage, L., Ravot, G., Jermutus, L., Minter, R. — 2008-04-14

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined. Parallel evolution strategies, with and without a recombination step, were carried out and both methods improved the affinity of an anti-Fas single chain variable fragment (scFv). The recombination step resulted in an increased population of affinity-improved variants. Moreover, the most improved variant, with a 22-fold affinity gain, emerged only from the recombination-based approach. An analysis of mutations preferentially selected in the recombined population demonstrated strong cooperative effects when tested in combination with other mutations but small, or even negative, effects on affinity when tested in isolation. These results underline the ability of combinatorial library approaches to explore very large regions of sequence space to find optimal solutions in antibody evolution studies.