Question

1. a)Give two characteristics of recombinant proteins cloned into the pRSET-FP plasmid.

b) pRSET is called an “expression vector”. Explain the function of the following in the context of producing the recombinant fluorescent protein.

T7 promoter:

RBS:

ATG:

6xHis:

c) What does “in frame” refers to?

Answer 1

Q1)

A)

• The insertion site for a recombinant protein cloned into the pRSET-FP plasmid is downstream of a N-Terminal Fusion Peptide encoding for a 6xHis Tag, which can be used for protein purification using a Nickel-NTA (Ni-NTA) column.
• The fusion peptide present in the backbone of pRSET-FP also includes an Enterokinase recognition site, which allows the removal of the 6xHis Tag from the recombinant protein after purification.

B)

An expression vector is a plasmid that can be used to drive the 'expression' or synthesis of the insert protein in the target host cell.

• T7 promoter:
  • The T7 promoter is a DNA sequence recognized by the T7 polymeraze in a specific manner. The T7 polymeraze is highly specific and will only transcribe sequences downstream of the T7 promoter. Transcripts generated by the T7 polymeraze are initiated within the T7 promoter sequence. The T7 promoter in pRSET-FP allows for specific and controlled transcription by the T7 polymeraze from the vector.
    
• RBS:
  • The Ribosome Binding Site or RBS is a sequence that the bacterial ribosome binds to, in order to initiate translation from the ATG site downstream.
    
• ATG:
  • The N-terminal fusion peptide includes an ATG sequence, which is the translation initiation site for the recombinant protein. The ATG codon codes for the Amino Acid Methionine.
    
• 6XHis:
  • The 6xHis sequence included in the backbone of the pRSET-FP vector is a Histidine Tag that is used for purification of the recombinant protein. The 6xHis tag has high affinity for metal ions and this property is utilized for purification of the protein synthesized in the bacterial cell.

C)

'In frame' refers to the sequence 'read' by the ribosome to synthesize any protein. There are 4 letters in the Nucleic Acid alphabet, ATGC in DNA and AUGC in RNA. In order to encode for the more numerous standard Amino Acids, groups of 3 Nucleic Acid bases are used by the Ribosome to determine which Amino Acid to add to the growing polypeptide chain. This grouping is known as a Codon.

When the Ribosome recognizes the first codon (ATG), all downstream sequence is read by the Ribosome in subsequent groups of 3 Nucleic Acid bases. Theoretically, for any given DNA sequence, six different 'reading frames' are possible, three in the forward direction and 3 in the reverse. However, all codons, aligned with the initial ATG are used by the Ribosome for determining the protein sequence and these codons are called 'in frame'.

For example in the sequence ATGGGCTACTGGGCAACT; the codons are ATG, GGC, TAC, TGG, GCA and ACT and the sequence following the G in ATG is called in-frame.