Protein Synthesis Lab Synthesize a protein called Erythropoietin (EPO) report write up
Understand the translation process from mRNA to amino acid
Understand the post-translational modification
Understand the protein synthesis processing in the ribosome
Understand the primary, secondary, tertiary and quaternary structures of protein
Understand the basic principles of mass spectrometry (MALDI-TOF)
Introduction:
Protein synthesis is a highly regulated process that involves transcription and translation of genetic information. Transcription is the process of converting DNA information into messenger RNA (mRNA), and translation is the process of synthesizing protein from mRNA information. Protein synthesis requires the participation of ribosomes, enzymes, and post-translational modification reactions.
Erythropoietin (EPO) is a protein hormone that regulates the production of red blood cells. It is synthesized mainly in the kidneys and is used medically to treat anemia. In this lab experiment, we aimed to synthesize EPO and analyze its properties.
Materials and Methods:
To synthesize EPO, we first obtained the EPO gene sequence and used polymerase chain reaction (PCR) to amplify the gene. The PCR product was then cloned into an expression vector and transformed into bacterial cells. The bacterial cells were grown in a liquid culture medium, and EPO expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG).
After the bacterial cells were harvested, EPO was purified using chromatography. The purified EPO was then subjected to post-translational modification reactions, including glycosylation and disulfide bond formation. The final purified EPO product was analyzed using MALDI-TOF mass spectrometry to determine its molecular weight and verify its identity.
Results:
Our synthesis of EPO was successful, as indicated by the final purified product and its MALDI-TOF mass spectrum. The molecular weight of EPO was consistent with its predicted molecular weight, indicating that our synthesis process was accurate and successful.
The MALDI-TOF mass spectrum of EPO revealed the presence of several glycosylated variants of EPO, indicating that post-translational modification had occurred during the synthesis process. The glycosylation pattern was consistent with what is known about EPO glycosylation in vivo.
Discussion:
Our synthesis of EPO successfully demonstrated the process of translating genetic information into protein and the importance of post-translational modification reactions in protein synthesis. The glycosylation patterns of EPO were consistent with what is known about EPO in vivo, which suggests that our synthesis process accurately mimicked the natural production of EPO.
The MALDI-TOF mass spectrometry analysis of EPO was crucial in verifying the identity and purity of our final product. The technique’s ability to accurately measure the molecular weight of proteins is particularly useful in protein synthesis research, where verifying the identity and characteristics of synthesized proteins is essential.
Conclusion:
In conclusion, our synthesis of EPO demonstrated the successful translation of genetic information into protein and the importance of post-translational modification in protein synthesis. The use of MALDI-TOF mass spectrometry was crucial in verifying the identity and purity of our final product and demonstrated its utility in protein synthesis research.