Manufacture and expression of large structural genes

Abstract

Illustrated is the preparation and expression of manufactured genes capable of directing synthesis of human immune and leukocyte interferons and of other biologically active proteinaceous products, which products differ from naturally-occurring forms in terms of the identity and / or relative position of one or more amino acids, and in terms of one or more biological and pharmacological properties but which substantially retain other such properties.

Description

[0001]This a continuation-in-part of U.S. patent application Ser. No. 06 / 375,494, filed May 6, 1982 and now abandoned.[0002]The present invention relates generally to the manipulation of genetic materials and, more particularly, to the manufacture of specific DNA sequences useful in recombinant procedures to secure the production of proteins of interest.[0003]Genetic materials may be broadly defined as those chemical substances which program for and guide the manufacture of constituents of cells and viruses and direct the responses of cells and viruses. A long chain polymeric substance known as deoxyribonucleic acid (DNA) comprises the genetic material of all living cells and viruses except for certain viruses which are programmed by ribonucleic acids (RNA). The repeating units in DNA polymers are four different nucleotides, each of which consists of either a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) bound to a deoxyribose sugar to which a phosphate group is ...

AI Technical Summary

Benefits of technology

"The present invention provides a novel and efficient method for synthesizing linear, double stranded DNA sequences of a length in excess of about 200 base pairs, which can include entire structural genes coding for polypeptides of interest. The method involves preparing two or more different, linear, double stranded DNA sequences of about 100 base pairs in length, each containing a unique recognition site for a restriction endonuclease. These sequences are then serially inserted into a selected assembly vector and biologically amplified in a host microorganism. The desired DNA sequence is then isolated and can be inserted in a different expression vector to direct expression of the polypeptide of interest. The invention also provides novel manufactured genes coding for various polypeptides, including human immune interferon and leukocyte interferon."

Problems solved by technology

While the foregoing generalized descriptions of published recombinant DNA methodologies may make the processes appear to be rather straightforward, easily performed and readily verified, it is actually the case that the DNA sequence manipulations involved are quite painstakingly difficult to perform and almost invariably characterized by very low yields of desired products.

As an example, the initial “preparation” of a gene for insertion into a vector to be used in transformation of a host microorganism can be an enormously difficult process, especially where the gene to be expressed is endogenous to a higher organism such as man.

One laborious procedure practiced in the art is the systematic cloning into recombinant plasmids of the total DNA genome of the “donor” cells, generating immense “libraries” of transformed cells carrying random DNA sequence fragments which must be individually tested for expression of a product of interest.

Such sequential additions are continued until a complete gene sequence is developed, with the total procedure being very time-consuming and highly inefficient.

The time-consuming characteristics of such methods for total gene synthesis are exemplified by reports that three months' work by at least four investigators was needed to perform the assembly of the two “short”, insulin genes previously referred to.

Further, while only relatively small quantities of any manufactured gene are needed for success of vector insertion, the above synthetic procedures have such poor overall yields (on the order of 20% per ligation) that the eventual isolation of even minute quantities of a selected short gene is by no means guaranteed with even the most scrupulous adherence to prescribed methods.

The maximum length gene which can be synthesized is clearly limited by the efficiency with which the individual short segments can be joined.

Since this relationship is expotential in nature, even a small increase in the yield per ligation reaction will result in a substantial increase in the length of the largest gene that may be synthesized.

Inefficiencies in the above-noted methodology are due in large part to the formation of undesired intermediate products.

Proposals for increasing synthetic efficiency have not been forthcoming and it was recently reported that, “With the methods now available, however, it is not economically practical to synthesize genes for peptides longer than about 30 amino acid units, and many clinically important proteins are much longer”.

The procedure, which the authors characterize as “rapid”, is reliably estimated to have consumed nearly a year's effort by five workers and the efficiency of the assembly strategy was clearly quite poor.

In this context, the absence of procedures for rapid and efficient total gene manufacture which would permit codon selection is seen to constitute an even more serious roadblock to advances in the art.

Despite the advantages in isolation of quantities of interferons which have been provided by recombinant DNA techniques to date, practitioners in this field have not been able to address the matter of preparation of synthetic polypeptide analogs of the interferons with any significant degree of success.

No means is readily available for the excision of a fragment of the subject gene and replacement with a fragment including the coding information for a variant polypeptide sequence.

Further, modification of the reported cDNA-derived and manufactured DNA sequences to vary codon usage is not an available “option”.

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