Jean-Luc Jestin

Improving the display of proteins on filamentous phage

In phage display technology, polypeptides are displayed on the surface of filamentous bacteriophage by genetic fusion to a coat protein. However, the fraction of phage particles bearing the fusion protein can be low. Here we found that we could improve the display of a protein (Stoffel fragment of Taq polymerase fused to the p3 protein of the phage) by mutation of the signal sequence and use of helper phage with a protease-cleavable coat protein. Over multiple rounds of infection, proteolysis and binding to an anti-Taq antibody, we were able to select strongly for display of the fusion protein (> 50-fold), and for mutations in the translation initiation region and in the signal sequence of the fusion. This suggests a general means of improving the display of proteins on phage.

An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell transmission.

Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver‐transplanted patients a challenging task. Virus‐specific therapeutic antibodies are generally safe and well‐tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy‐chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage‐display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture–derived particles by interfering with the E2‐CD81 interaction. In contrast to some of the most broadly neutralizing human anti‐E2 monoclonal antibodies, D03 efficiently inhibited HCV cell‐to‐cell transmission. Conclusion: This is the first description of a potent and broadly neutralizing HCV‐specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell‐to‐cell transmission. (Hepatology 2013;53:932–939)

From the analysis of protein complexes to proteome-wide linkage maps

Recent advances in genomics have led to the accumulation of an unprecedented amount of data about genes. Proteins, not genes, however, sustain function. The traditional approach to protein function analysis has been the design of smart genetic assays and powerful purification protocols to address very specific questions concerning cellular mechanisms. Lately, a number of proteome-wide functional strategies have emerged, giving rise to a new field in biology, proteomics, that addresses the biology of a cell as a whole.

Functional cloning by phage display

This review focusses on the isolation of proteins from genomic or cDNA expression products libraries displayed on phage. The use of phage display is highlighted for the characterization of binding proteins with diverse biological functions. Phage display is compared with another strategy, the yeast two-hybrid method. The combination of both strategies is especially powerful to eliminate false positives and to get information on the biochemical functions of proteins.

The genetic code and its optimization for kinetic energy conservation in polypeptide chains

Why is the genetic code the way it is? Concepts from fields as diverse as molecular evolution, classical chemistry, biochemistry and metabolism have been used to define selection pressures most likely to be involved in the shaping of the genetic code. Here minimization of kinetic energy disturbances during protein evolution by mutation allows an optimization of the genetic code to be highlighted. The quadratic forms corresponding to the kinetic energy term are considered over the field of rational numbers. Arguments are given to support the introduction of notions from basic number theory within this context. The observations found to be consistent with this minimization are statistically significant. The genetic code may well have been optimized according to energetic criteria so as to improve folding and dynamic properties of polypeptide chains.

Efficient display of two enzymes on filamentous phage using an improved signal sequence

Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA. In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA. Optimization of this link can be achieved by adjusting the signal sequence of the fusion. In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50-fold higher incorporation of fusion proteins in phage particles. In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide. Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins. These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.

A rationale for the symmetries by base substitutions of degeneracy in the genetic code.

The first symmetry by base substitutions of degeneracy in the genetic code was described by Rumer (1966) and the other symmetries were identified later by Jestin (2006) and Jestin and Soulé (2007). Here, a rationale accounting for these symmetries is reported. The number of non-synonymous substitutions over the replicated coding sequence is written as a function of the substitution matrix, whose elements are the number of substitutions from any codon to any other codon. The p-adic distance used as a similarity measure and applied to this matrix is shown to be biologically relevant. The rationale indicates that symmetries by base substitutions of degeneracy in the genetic code are symmetries of the measures of the number of non-synonymous substitutions for sets of synonymous codons.

A method to predict edge strands in beta-sheets from protein sequences

There is a need for rules allowing three-dimensional structure information to be derived from protein sequences. In this work, consideration of an elementary protein folding step allows protein sub-sequences which optimize folding to be derived for any given protein sequence. Classical mechanics applied to this system and the energy conservation law during the elementary folding step yields an equation whose solutions are taken over the field of rational numbers. This formalism is applied to beta-sheets containing two edge strands and at least two central strands. The number of protein sub-sequences optimized for folding per amino acid in beta-strands is shown in particular to predict edge strands from protein sequences. Topological information on beta-strands and loops connecting them is derived for protein sequences with a prediction accuracy of 75%. The statistical significance of the finding is given. Applications in protein structure prediction are envisioned such as for the quality assessment of protein structure models.

Streptococcus agalactiae DNA polymerase I is an efficient reverse transcriptase

Genome annotations result generally from large sequence alignments by bioinformatics. Large scale biochemical data are more difficult to obtain. They derive for example from directed protein evolution experiments by selection. A previously reported directed enzyme evolution experiment by in vitro selection of Stoffel fragment variants of Taq DNA polymerase I was used here to predict the activities of Streptococcus agalactiae DNA polymerase I. The reverse transcriptase activity of S. agalactiae DNA polymerase I was measured and the kinetic parameters for this RNA-dependent DNA polymerase are given. RNA-templated DNA repair is suggested as a possible biological function for this biochemical activity.

Characterisation of a DNA Polymerase Highly Mutated Along the Template Binding Interface

Phage display establishes a link between a polypeptide and its corresponding gene. It has been much used for the isolation of proteins binding to chosen molecular targets. A second link was designed more recently between a phage-displayed enzyme and its reaction product. Affinity chromatography for the product then allows the isolation of catalytically active enzymes and of their genes. Using this strategy, a polymerase with 15 mutations was selected by directed evolution of Thermus aquaticus DNA polymerase I. The kinetic characterisation reported here highlights the variant’s broad template specificity and classifies this enzyme as a thermostable DNA-dependent and RNA-dependent DNA-polymerase.