cDNA Phage Library Construction Service

Alpha Lifetech Inc. can offer native, semi-synthesized, and fully synthesized peptide libraries based on phage display technology. Our experts have extensive experience in library construction, including chemical peptide libraries, phage display peptide libraries (such as substrate peptide libraries), antibody libraries, and related libraries.


Phage Display Library


In the combinatorial molecular biology technique known as phage display, libraries of molecules—typically peptides and proteins—are fused to ready-made bacteriophage scaffolds. The method has advantages over others based on tiny compounds due to the phage's survivability. The libraries are easily amplified by infecting bacteria, and since the virion contains the phage genome, DNA sequencing can quickly deconvolute the libraries. Additionally, libraries with orders of magnitude more members than those employing tiny molecules can create since the production of degenerate DNA regulates variety. Additionally, phage display libraries may be created in any lab using basic microbiology equipment, making it much less expensive than the creation of small-molecule libraries. Given its strength, flexibility, convenience, and minimal expense, phage display has been broadly utilized and integrated into different applications.


cDNA Phage Display Library


cDNA is the complementary DNA, produced after the reverse transcription of RNA by the enzyme reverse transcriptase. cDNA library is the collection of host cells bearing cloned cDNA fragments that constitute a part of the transcriptome of an organism. cDNA libraries are maintained to have the clone of expressed genes of an organism, as the cDNAs are produced from the fully expressed mRNAs. Mature mRNA undergoes post-transcriptional modifications like splicing, where the non-coding regions like introns are removed so the cDNA won't contain the codes for the intron region.

For the synthesis of cDNA from mRNA, a poly-A tail of mRNA is used as a priming site, a short tag of oligo dT with a free 3'OH group will bind and be extended by reverse transcriptase to create cDNA. The next step is to remove the mRNA, which is achieved by treating it with RNase enzyme, resulting in the single-stranded cDNA. The single-stranded cDNA needs to be converted to double-stranded cDNA with the help of DNA polymerase. The single-stranded cDNA itself provides the free 3'OH group for polymerase extension by forming a hairpin loop-like structure, which can later be cleaved using a nuclease. Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids. The cloned bacteria are then selected, commonly using antibiotic selection. Once selected, the library is produced, which can later be grown and sequenced to compile the cDNA library.



Fig 1 cDNA library construction


High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation.

Phage cDNA display library construction technology first reverse-transcripted mRNA extracted from tissues or cells into cDNA fragments and then inserted into carrier genes to make the phage surface express various proteins encoded by the cDNA library in the form of fusion proteins. The steric hindrance is minor so that it can maintain a relatively independent spatial structure and biological activity. Because the composition of the cDNA library is more complex, the proteins displayed on the phage surface are more diverse, which increases the possibility of screening with specific ligands to target molecules with more potent binding forces. 

Alpha Lifetech Inc. can offer scientists three different cDNA libraries based on our phage display technology. These libraries include:


M13 Phage Display cDNA Library


 We can use M13 phage to construct cDNA display libraries.


Fig 2 M13 Phage Display Library Construction 


Since the cDNAs must be in the same reading frame as the phage coat protein and when fused to the N terminus, they cannot contain in-frame stop codons that would cause the display of the fusion protein to end prematurely, displaying cDNA libraries on filamentous phage has proven to be complicated. The significant variation among the polypeptide sequences that must form functional fusions with the phage coat protein further complicates the cDNA library presentation. To solve these problems, many other display strategies have been created, including fusion to the C terminus of pVI, indirect display on pIII utilizing a c-fos/c-jun attachment, and cDNA fragmentation.


T4 Phage Display cDNA Library


In several ways, bacteriophage T4 differs from bacteriophage M13. The double-stranded DNA (dsDNA) genome of T4 is substantially bigger, has a tail structure, and codes for 50 distinct proteins. T4 phage is composed of three sections, which may be distinguished based on appearance:

-- a head.

-- a fistulous and contractile neck for DNA injection.

-- a tail portion for identifying and attaching to bacteria's surface.


Three coat proteins—gp23, gp24, and gp20—make up the majority of the icosahedral shape of the viral head, where T4 protects its DNA. Two non-essential proteins, HOC and SOC, are also present and are coated on the icosahedral head, among other coat proteins. Additionally, unlike M13, T4 only travels through the lytic lifecycle, which means that upon infection, offspring phage particles are assembled in the cytoplasm and released by the lysis of the host bacteria.

The T4 phage is often used to study complex proteins that E. Coli cannot secrete because the assembly of the virus particles takes place inside the host cell, thus displaying multiple peptides or proteins without the need for a secretory pathway. In addition, there are two binding sites on the capsid protein of T4, namely SOC site and HOC site. Therefore, the phage can fuse two foreign peptides/proteins with different properties and display them on its surface at the same time, and the number of copies displayed is also significant.

Generally speaking, T4 phage display systems can provide significant benefits over other display methods:

-- Greater genome DNA allows for greater insertions.

-- high display density.

-- Dual display: Separate displays of two distinct molecules on HOC and SOC.

-- Available with both N- and C-terminal insertion.

-- No need to secrete from the membrane, averting host toxicity and conformational alterations.

-- In addition to E. coli, there are other host bacteria strains.


fig 3 structure of tPhage


T4 phage display cDNA library has many uses in the production of antibodies. In particular, the production of multicomponent vaccines has shown significant promise for T4 phage display in a variety of scientific and medicinal domains. When a traditional M13 phage display is unsuccessful, it offers a potential substitute.


T7 Phage Display cDNA Library


The lambda and T7 capsids are formed in the reducing environment of the bacterial cytoplasm and do not require the proteins to be translocated across membranes, in contrast to filamentous phage display, which necessitates protein assembly in the periplasmic space, an oxidizing environment that, for example, favors the formation of disulfide bridges.

Coat protein 10 is linked at its C-terminus to cDNA expression products in T7 phage display. This system had a large user base.


fig 4 tPhage Display Library Construction


In vitro evolution of antibodies and antibody surrogates in the form of randomized fragments on various scaffold proteins, the discovery of enzyme substrates and inhibitors, identification of functionally coupled proteins and analysis of protein-protein interactions, design of catalytic antibodies (abzymes) and enzymes with novel speciation were all accomplished using phage display, which frequently required creative modifications. In chromatographic procedures and biosensing, protein ligands chosen from phage libraries as well as complete recombinant phage have been employed as affinity matrices.

Alpha Lifetech Inc. is able to display whole cDNA products on the surface of the proper phage system thanks to our sophisticated phage display technology. Our high-quality libraries may achieve greater capacity, density, and proper orientation compared to ordinary cDNA libraries, which furthers the examination of numerous protein candidates. Additionally, several cDNA library types, such as standard libraries, full-length libraries, normalized libraries, and subtractive libraries, can be customized to meet the needs of our clients. For more details and to receive a customized quotation, please feel free to contact us.