Heterolog expression av proteiner

Heterolog expression av proteiner

Expression av protein i icke-naturlig (“främmande” värdorganism)

Fördelar:

• Större mängder (—> ekonomi, renhet)

• Homogen genpool (värdorganism är klon)

• Tillgång (från annars svåra källor)

• Etik säkerhet

• möjlighet till strukturell modifiering

  • stabilitet

  • reningsmetodik

  • funktion (ex funktion som kan få ytterligare fördelar)

Vilka krav behövs för att genomföra heterolog expression av proteiner?

Krav:

• Tillgång till gen eller cDNA

  • nödvändig utgångsmaterial

  • från bakterie —> använd gen

  • från eukaryot organism —> använd cDNA

• expressionssystem (E. coli, jästcell osv)

• Analysverktyg metoder

  • Vi måste veta att vi fått proteinet av intresse.

Step by Step synthesize cDNA

Mitt svar:

• mRNA har alltid 3’ polyA svans som endast innehåller adenosin.

• First strand synthesis:

  • Adderade oligo(dT) primers (12-20 tymin nukleotider) hybridiserar till 3’ polyA svans till varje mRNA.

  • Reverse transkriptas binder till primer regioner och syntetiserar komplementär sträng mha adderade dNTPs

  • mRNA degraderas med alkali (gör lösningen basisk —> deprotonerar OH-grupp —> mRNA degraderar. Finns också enzym som degraderar mRNA)'

• Second strand synthesis

  • oligonucleotide of known sequence is often ligated to the 3’ end of the cDNA

  • DNA polymerase I (adderad) och dNTPs (adderad) förlänger primern för att ge dubbelsträngat cDNA

• Amplification

  • Görs med PCR ofta

  • möjlighet att modifiera 5’-ändarna på oligonukleotiderna —> modifiera\optimera gen\cDNA sekvens

  • Värme separerar strängarna

  • Taq DNA polymeras läggs till

Chattens svar:

Process of creating complementary DNA (cDNA) from messenger RNA (mRNA) using reverse transcriptase enzyme. Involves three main steps: mRNA isolation, reverse transcription, and cDNA amplification. Allows study of gene expression and analysis of specific genes.

Step-by-step breakdown of the process of creating complementary DNA (cDNA) from messenger RNA (mRNA) using reverse transcriptase enzyme:

1. mRNA isolation: Extract mRNA molecules from the cells or tissues of interest. This can be done through methods like cell lysis and separation of mRNA from other cellular components.

2. Reverse transcription: Use reverse transcriptase enzyme, derived from retroviruses, to synthesize cDNA using the mRNA as a template. Reverse transcriptase synthesizes cDNA in the opposite direction of mRNA transcription.

3. cDNA amplification: Amplify the cDNA to ensure there is enough for further analysis. Polymerase chain reaction (PCR) is commonly used for cDNA amplification. PCR utilizes specific primers to initiate DNA replication and exponentially amplify the cDNA.

By following these three steps, researchers can convert mRNA into stable cDNA, allowing them to study gene expression, identify differentially expressed genes, and gain insights into cellular processes. The generated cDNA can also be used for cloning, gene expression analysis, and other applications in biomedical research.

Expressionssystem

Beroende på vad man vill uttrycka och hur komplext det är ska man välja värdcell.

• Genetisk reglering

  • Promotor

  • terminator

  • signalsekvens?

  • translationssekvenser?

• Post-translationell modifiering? krav för funktion?

  • Ej säkert att modifieringar gener proteiner normalt hade gått igenom behövs för vår biotekniska applikation.

Värdcell:

• Högt utbyte —> bakterier alt jäst

  • De växer snabbt

  • man kan manipulera dem att satsa på produktion av vårt protein.

• posttranslationella modifieringar —> eukaryota system

  • Jäst - risk för hyperglykolysering

  • insekter

  • mammalieceller (HeLa, cos, CHO, m.m)

  • hela djur - extrema fall, etiskt svårt

Expressionssystem

Promotor

• ex T7, Tac,

• inducerbar (IPTG, LacI)

Ribosom binding (shine-dalgarno box)

• Viktigt för att ribosomens (undre del ??) ska binda

Selektionsmarkör (antibiotikaresistent, fluorescens)

Gene encoding repressor that binds operator and regulares bacterial promoter

Transcriptiontermination sequence

A vector plasmid typically contains an origin of replication, a selectable marker, and a cloning site for inserting foreign DNA.

A vector plasmid typically contains an origin of replication, a selectable marker, and a cloning site for inserting foreign DNA.A vector plasmid, an essential tool in molecular biology research, encompasses several crucial elements that enable its functionality and versatility. At its core, a vector plasmid serves as a carrier for foreign DNA, allowing scientists to manipulate and study specific genetic material. While the original statement encapsulates the fundamental components of a vector plasmid, let's delve deeper into each element to gain a comprehensive understanding.

Firstly, an origin of replication (ori) is an indispensable component of a vector plasmid. This specific DNA sequence serves as the starting point for DNA replication, allowing the plasmid to be autonomously replicated within a host organism, such as bacteria or yeast. The ori ensures that the vector plasmid can be efficiently reproduced, providing an abundant supply for further experimentation and analysis.

Secondly, a selectable marker plays a pivotal role in distinguishing cells that have successfully taken up the vector plasmid from those that haven't. This marker, often a gene encoding resistance to an antibiotic or toxin, allows researchers to selectively grow only the transformed cells that carry the vector plasmid. By incorporating a selectable marker, scientists can ensure that the desired genetic material is propagated while non-transformed cells are eliminated, streamlining the process of identifying and studying the target DNA.

Furthermore, a vector plasmid includes a cloning site, also known as a multiple cloning site (MCS) or a polylinker. This region contains a series of unique restriction enzyme recognition sites, enabling the insertion of foreign DNA fragments with precision. These restriction sites act as molecular scissors, cleaving the vector plasmid at specific locations, which allows the foreign DNA to be seamlessly integrated. The MCS provides flexibility and convenience, allowing researchers to insert various DNA fragments of interest into the vector plasmid, facilitating the study of specific genes or regulatory elements.

In conclusion, a vector plasmid encompasses an origin of replication, a selectable marker, and a cloning site, which collectively enable the successful manipulation and study of foreign DNA. The ori ensures autonomous replication of the plasmid, the selectable marker facilitates the identification of transformed cells, and the cloning site allows for the precise insertion of DNA fragments. By understanding the intricacies of these components, scientists can harness the power of vector plasmids to advance our knowledge of genetics and molecular biology.

Fördelar med expressionssystem

• möjlighet att modifiera

  • taggar: Effektiviserar rening (ex his-tag\IMAC, GST, MBP)

  • Detektion (immunotaggar, fluorescens (GFP-fusion))

  • löslighet (proteinet fälls ej ut och ex bryts ner)

    • ordlingstemperatur

    • Co-uttryck av chaperonproteiner

    • fusionsproteiner