mRNA-peptide fusion

口述:張文章 老師

整理:陳郁鴻

 

mRNA-peptide fusion

利用linkage每一proteinencode其之DNA sequence(例如mRNA-peptide fusion),做為在in vitro製備nucleic acid-encoded peptideprotein libraries有效率的方法。

 

    在技術方面的考量有別於ribosome display,因為ribosome display過程中可能遭遇ribosome-mRNA-peptide複合體較不穩定的問題,不易於selection時維持其複合體的完整性,因此以peptidemRNAcovalent linkage的方法在技術上較具優勢。藉由一種抗生素-puromycin,為aminoacyl-tRNA analoguecovalent linkage

 


    Puromycin分子依附於DNA spacer接在mRNA 3’-end,於in vitro translation時當ribosome停於DNA spacer,末端的puromycinaminoacyl-tRNA analogue則進入ribosomeA site並與peptide產生linkage,此法亦稱mRNA display 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

圖(Figure 7.)顯示典型的mRNA display實驗:

首先將DNA libraryin vitro轉錄出mRNA library

合成含puromycin3’-end30-bp DNA sequence,並接合至total mRNA

mRNA-DNA-puromycin hybrid molecular進行in vitro translation

得以puromycin共價結合之mRNA-peptide複合體

再經isolationreverse transcription,和利用specific antigenselection

篩選到的複合體進行PCR放大,可用作定序或轉譯等進行其他實驗

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

                                                                                                Figure 1mRNA-protein fusion synthesis

 

 

(a)     雙股DNA模板於in vitro轉錄mRNA模板

(b)    biotinylated puromycin-linker作用

(c)     固定於neutravidin beads

(d)    washing並除去unbound mRNA

(e)     beads照射UV light

(f)      elutephoto-ligated mRNA-puromycin template以用來進行in vitro translation構成mRNA-protein fusions library

 

T7transcription promoter sequence

 

TMVa portion of the tobacco mosaic virus 5’-UTR with good initiation codon context

 

random 10 Fn3a sequence coding for a randomized 10th fibronection type III domain

 

consta constant sequence which codes for the linker hybridization and crosslinking sites as detailed in Fig. 2

Pupuromycin

 

Psopsoralen

 

PC-biotina photo-cleavable biotin-tag described in Fig. 2.


 

 

 

Figure 2:設計之mRNA 3’-end模板和puromycin-linker序列

 

mRNA-peptide fusion方法的缺點

#所有步驟皆是in vitro情況下進行

#對membrane proteins或必須post-translational modificationproteins則不易操作

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

參考資料

Discussion on the paper: H. Lin and V.W. Cornish.  Screening and Selection Methods for Large-Scale Analysis of Protein Function Angew. Chem. Int. Ed., 41, 4402- 4425 (2002).

 

http://mirror.ensta.fr/molecules/papers/51201259.pdf