Methods for improving recombinant protein expression

Medicilon is a leading provider of comprehensive, high quality recombinant protein and bioprocess services. We offer a variety of recombinant protein expression platforms along with a host of other protein services like chemical protein synthesis, protein refolding and structural biology services.Email:marketing@medicilon.com.cn Web:www.medicilon.com

Materials and methods are provided which allowed for increased expression of a transfected gene of interest in a recombinant host cell.

This application claims the benefit of priority to U.S. Provisional Application Serial No. 61/231,906 filed August 6, 2009, entitled "Methods for Improving Recombinant Protein Expression," Attorney Docket No. 31351/44743, the entire content of which is incorporated by reference herein.

Field of Invention:This invention has practical application in the field of recombinant protein expression in eukaryotic cells by means of increasing selection pressure on a vector thereby increasing vector-associated heterologous protein expression.

Background:In the field of recombinant protein production, increasing expression of a transfect gene is a fundamental priority during cell line development. Improving transcription, translation, protein folding and secretion are all targets of intense research to increase titers of the heterologous protein.

Regardless of methods used in the past, there exists a need in the art to provide better methods for recombinant protein production that increase yield of the desired protein.

Summary of the Invention:In one aspect the invention provides a method for increasing heterologous protein expression in a host cell comprising the steps of culturing the host cell comprising a first heterologous polynucleotide sequence encoding the heterologous protein under conditions that allow for protein expression, the first polynucleotide encoded on a vector, the host cell further comprising a second polynucleotide sequence having a protein coding sequence for a selectable marker protein, the second polynucleotide having a sequence modification compared to a wild-type polynucleotide encoding the selectable marker protein, the sequence modification reducing translation efficiency of mRNA encoded by the second polynucleotide, the second polynucleotide having the sequence modification and the wild-type polynucleotide encoding identical amino acid sequences for the selectable marker protein. In one aspect, the first polynucleotide and the second polynucleotide are in a single vector, and in one embodiment of this aspect, the first polynucleotide and second polynucleotide are each under transcriptional control of distinct promoters. In other aspects, the first polynucleotide and the second polynucleotide are in separate vectors. In yet another aspect, the first polynucleotide and second polynucleotide are under transcriptional control of the same promoter.

In one embodiment of the method, the modification is in an untranslated region of the second polynucleotide encoding the selectable marker protein, and in certain aspects, the modification is in a 5' untranslated region and/or the modification is in a 3' untranslated region.

In another embodiment of the method, the modification is in a protein coding region of the gene encoding the selectable marker protein. In one aspect, the modification is within 25, 20, 15, 10, or 5 codons of an initiating codon of the protein coding region for the selectable marker gene coding sequence.

In another aspect of the method, the protein coding sequence in the second polynucleotide sequence comprises at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding the selectable marker protein, the modified codon being a codon that is not a preferred codon for the encoded amino acid for the host cell. In one aspect, the protein coding sequence in the second polynucleotide sequence comprises at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding the selectable marker protein, the modified codon being a codon that is a least preferred codon for the encoded amino acid for the host cell.

In another aspect of the method, the protein coding sequence in the second polynucleotide sequence comprises at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding the selectable marker protein, and the modification introduces a change in secondary structure of the mRNA which reduces translation efficiency of the mRNA. In one embodiment of the method, the protein coding sequence in the second polynucleotide sequence comprises at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding the selectable marker protein, and the modification increases codon pairing in the mRNA.