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US 8,527,211 B2Granted
Method to produce recombinant MBP8298 and other polypeptides by nucleotide structure optimization
OwnerReza Zareie, Mahdi Abbasianinventors
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Abstract
A method for recombinant production of a polypeptide, exemplified by the therapeutic peptide MBP8298, by recombining and optimizing the nucleic acid encoding said polypeptide, expressing said nucleic acid in a microbial host cell, isolating said polypeptide from the host, and releasing embedded polypeptide from fusion partners or peptide concatamers are explained. Such method may provide increased production or simplified downstream processing for the polypeptide of interest.
Claims7
A method for predicting the expression efficiency for recombinant production of a protein by prokaryotic cells, the method comprising the steps of:
- a) providing a mRNA sequence which codes for the protein to be produced, wherein said mRNA includes a translation initiation region (TIR);
- b) predicting a secondary structures for a 5′ region of said mRNA by a computer program executed on a computer;
- c) estimating the probability of said mRNA structure;
- d) calculating the fraction of unfolded TIR for said mRNA structure according to:
- F
- u
- =
- 1
- exp
-
- (
- -
- δ
-
-
- G
- TIR
- _
- Tk
- )
- +
- 1
- in which Fu is fraction of unfolded TIR, δGTIR is free energy of TIR (kcal/mole), T is the absolute temperature and k is the Boltzmann constant (0.001986266 kcal/mole/degree K);
- e) calculating probability of unfolded TIR in said mRNA sequence based on:
- Pu=PS×Fu
- in which Pu is probability of unfolded TIR, PS is said probability of mRNA structure (of step c) and Fu is said fraction of unfolded TIR for said structure of step d;
- and
- linking said probability of unfolded TIR (of step e) with predetermined expression Efficiencies.
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Dependencies are inferred from the claim text.