Methods to screen peptide display libraries using minicell display

Abstract

A minicell display method has been developed which has significant advantages for screening peptide libraries for candidates that can bind and effectively modulate a particular biological process. The method, based on the small, anucleate minicell, has increased versatility in generating unique sequences to screen as well as increasing the size of the peptides to be screened. In vivo mutagenesis, at the level of protein synthesis, as well as DNA replication, increases diversification of the library to be screened and therefore substantially increases the number of potential peptides that can modulate a particular biological response or mechanism.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] Priority is claimed to U.S. Provisional Application Ser. No. 60 / 274,039 filed on Mar. 7, 2001, and U.S. Provisional Application Ser. No. 60 / 306,946 filed on Jul. 20, 2001.FIELD OF THE INVENTION [0002] The present invention is generally in the field of high throughput peptide screening, and in particular relates to a minicell display technology for generation and screening of random peptides. BACKGROUND OF THE INVENTION [0003] The interaction between cognate proteins in receptor-ligand complexes, enzyme substrate reactions and antibody-antigen binding reactions has furthered the understanding of the molecular interactions required to effect a response in a wide range of processes. The search for new peptide molecules which can bind to selected targets and effectively modulate a particular biological process is at the forefront of agricultural, biological, and medicinal research. [0004] There are several examples of methods that use pepti...

AI Technical Summary

Benefits of technology

[0021] In certain embodiments, it is desirable that the first gene encodes an outer membrane protein, or portion thereof, amenable to fusing large oligonucleotides encoding proteins greater than 302 amino acids in length. The 17K antigen of Rickettsia rickettsii is preferred. In one embodiment the expression of the fusion protein is regulated by an inducible DNA regulatory element, for example, a lac promoter, tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), trp promoter, or lacUV5 promoter. Other suitable microbial promoters may be used as well. By using an inducible promoter, the oligonucleotide fusion will remain quiescent until the addition of the inducer. This allows control of the timing of production of the gene product.

[0023] Optionally, the method consists of transforming suitable host minicell strains exhibiting a mutator phenotype and subsequent induction of the minicells to replicate acquired plasmid DNA. The method further includes generating a bacterial minicell strain exhibiting mutator phenotype. Mutations in genes responsible for DNA repair typically have a mutator phenotype. For example, mutations in the genes responsible for the methyl-directed mismatch repair of DNA, designated mutS, mutL, and mutH, increase the spontaneous mutation frequency about 1000-fold. Incorporating one, two, or all three of these mutations into the parent bacterial cell results in the in vivo diversification of the peptide display library within the anucleate minicell population.

[0027] Those minicells that bind to the target molecule are separated from those that do not. Optionally, the peptides displayed on the minicells may be labeled with molecules or compounds such as radioactive isotopes, rhodamine, or FITC before, during, or after expression of the display library. This serves to facilitate subsequent identification of the bound peptide of interest. For example, antibodies available to the target molecule may be used to immunoprecipitate the interacting complex. If the interacting peptide is radiolabeled, the complex can be easily distinguished and visualized by autoradiography, a method well established within the art. Optionally, the minicells may be supplemented exogenously with amino acid analogs to be incorporated into the peptide being synthesized in vivo.

[0030] The bound minicells can be easily eluted from the target molecule and the peptide encoding expression vectors isolated to extract information. The DNA sequence of the peptide, DNA base composition, the molecular weight, and / or whether any secondary structures exist within the sequence can then be determined.

Problems solved by technology

However, in most cases, the foreign protein interferes with localization, and thus, the fusion protein is unable to reach the cell surface.

Although the phage and bacterial display systems have provided unique routes to elucidating new peptides which can bind target molecules with new or enhanced binding properties, there are several important limitations that need to be considered.

Problems arise when larger peptide inserts (more than 100 amino acids) disrupt the function of the coat protein and therefore phage assembly.

Insert size constraints affect the applicability of these systems as well.

A limitation of the phage and bacterial display systems resides in the inability of these systems to incorporate amino acid analogs into peptide libraries in vivo.

In vivo, amino acid analogs disrupt the cellular machinery used to incorporate natural amino acids into essential proteins as well as the growing peptide chain of interest.

Technically cumbersome protocols can be time consuming when attempting the in vitro translation methods frequently used to incorporate amino acid analogs into a peptide sequence.

Peptides that are toxic to the bacterial cell and therefore lethal cannot be screened for in phage or bacterial display systems.

Phage and bacterial display also rely upon cumbersome and time consuming techniques in order to keep conditions optimal for cell growth and cell viability.

Infecting bacterial cells, harvesting the phage, and re-infecting several rounds is very time consuming.

Although the selection of specific mutations to be introduced into the gene is usually based on published reports describing the effects of the mutations on the activity or function of other homologous proteins, it is still difficult to predict the affect of the mutation or substitution.

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