DNA Cloning and Expression of HMGB1 in E. Coli

Liam Arnade-Colwill, Carbone/Yang

Background on HMGB1

• HMGB1 is released by damaged mesothelial cells in response to asbestos exposure

• Initiates the inflammatory response that can lead to mesotheliomadevelopment

Project Objectives

• To express HMGB1 in E. Coli in order to obtain enough samples of the protein to conduct further laboratory experiments investigating the link between HMGB1 and mesothelioma

• To learn important tools and techniques to work with protein and DNA in the future and to gain invaluable laboratory experience

Project Outline

• Phase One: DNA cloning

– Design gene-of-interest

– Ligate into PGEMT plasmid

– Transformation of DH5-a cells

– Screening

– Cell Growth

– Plasmid Extraction

– Restriction Digestion

• Phase Two: Expression

– Ligate GOI into PET14B expression vector

– Transformation of BL21 cells

– Cell Growth

– Induction of gene of expression with IPTG

– Protein purification

Protein Expression

• DNA is transcribed by RNA polymerase in the nucleus to form pre-mRNA

• Pre-mRNA is processed to form mRNA• mRNA leaves the nucleus and binds to ribosomes,

beginning translation• tRNA builds a polypeptide chain (protein)

DNA

Transcription

mRNA

Translation

Protein

Factors that affect Gene Expression

Rare Codons in HMGB1 Sequence

• 25/216 codons are rare for E. Coli

DNA Cloning

• 1) Add “A” overhangs to the the 3’ ends of our gene block

• 2) Ligate our gene-of-interest into PGEMT plasmids

• 3) Induce transformation of DH5-alpha cells through heath shock

• 4) Allow cell culture to grow on LB-Amp plates

Advantages of the PGEMT Vector

• “Copy Number” refers to the ability of DNA to replicate

• PGEMT is a high copy number plasmid, which means it yields a large amount of DNA

Blue/White Screening

• If gene-of-interest is absent from PGEMT, the lacz gene is activated and a blue color is expressed

• If the gene-of-interest is present in PGEMT, the lacz gene is inactivated and blue is no longer expressed

Colony PCR

• If gene-of-interest is present in PGEMT, a longer band of DNA is amplified, resulting in a single band of DNA

• If gene-of-interest is also absent, small and large bands of DNA are amplified, resulting in multiple bands of DNA

L 1 2 3 4 5 6 7 8

700 bp

Growth, Plasmid Extraction and Restriction Digestion

• After isolating the colonies with recombinant plasmids, we prepared a glycerol stock of our cells to grow them and make multiple copies of our gene

• Next, we performed plasmid extraction to re-obtain our PGEMT vector

• From there, we performed restriction digestion and ran a gel to isolate our gene-of-interest, then conducted gel extraction to obtain a pure sample of our replicated DNA

Expression

• 1) Ligate replicated DNA into PET14B expression vector

• 2) Induce transformation of BL21 cells through heat shock

• 3) Induce expression through addition of IPTG

Advantages of BL21 and PET14B

• HMGB1 can be toxic in high quantities to E. Coli, resulting in cell death

• PET14B is a low copy number plasmid, meaning that is does not replicate as easily. This results in less protein expression

• BL21 is protease deficient, protecting protein

• PLysS, a plasmid found in BL21, blocks gene expression until the addition of IPGT to sustain the viability of the cell

Results/Conclusion

• Five strong bands of protein in induced samples, no band of desired protein in un-induced sample (negative control)

• Protein is 25kDa, which indicates it is HMGB1

IPTG - + + + + +

25kDa

Next Steps

• Re-induce expression of HMGB1 in BL21 cells with IPTG

• Extract protein through protein purification process

• Quantify data with micro-plate system

Acknowledgments

• Dr. Carbone

• Dr. Yang

• Sandra Pastorino

• Vishal Negi

• Karin Koga

• All the other interns!

• UH Cancer Center