DD294279A5 - PROCESS FOR THE PREPARATION OF PROTEINS AND POLYPEPTIDES - Google Patents

Abstract

The invention in the field of biotechnology relates to a process for the preparation of such yeast-foreign proteins, etc., which are to be stored in vacuoles of the cells of a host strain of S. cerevisiae in high concentration. For this, a C-terminal amino acid sequence serving as a vacuole transport signal sequence is isolated from a known plant storage globulin approximately from Vicia faba, which is fused to the C-terminal amino acid sequence of the protein to be stored, whereby almost the entire synthesized gene products be stored in the vacuoles. {biotechnology; genetic engineering; Protein; Yeast; Host strain; Signal sequence; Memory globulin; gene}

Description

Field of application of the invention

The invention relates to a process for the preparation of proteins and polypeptides according to the preamble dos claim. 1

Characteristic of the prior art

Such a method of synthesizing yeast-foreign proteins or polypeptides, especially - but not only - for their secretion into a culture fluid, is already known from a proposal of the applicant; It has also proven itself in extracellular secretion.

However, if it is desired to ensure that the synthesized proteins or polypeptides are instead stored in the vacuoles of the yeast cells, it soon becomes apparent that an improvement of the method is desirable. In fact, in the case of the N-terminal fusion of the two amino acid signal sequences there is the danger that active sites of the proteins or polypeptides are destroyed; Relatively long and hence unstable amino acid signal sequences are also required for this particular application.

Object of the invention

By means of the invention, the synthesis of yeast-free protein in a host strain of the type described in greater detail is made possible with high intracellular concentration.

Explanation of the essence of the invention

Accordingly, the object of the invention is to avoid the disadvantages of the known process and to modify it so as to preserve its advantageous effects.

According to the invention, the object is achieved in that the vacuolar transport signal sequence derived from the plant storage globulin comprises a C-terminal amino acid sequence of at least 76 amino acids, which is fused to the C-terminal amino acid sequence of the protein or polypeptides to be incorporated into the cell. Surprisingly, it has been found that at least two different amino acid signal sequences can be extracted from the plant storage globulin for very different applications: an N-terminal amino acid sequence, as described above for controlling extracellular secretion, and according to the invention a C-terminal amino acid sequence, but now a vacuole transport signal sequence useful for storing the gene products in the cell. It is particularly advantageous if the vacuole transport signal sequence is obtained from legumin, in particular from the legumin B subunit of Vicia faba.

The method is very suitable for a construct using the structural gene of a hydrolase, such as that of chymosin, or an inverase.

The invention overcomes the disadvantages described above, and the synthesized gene products are completely, i. H. more than 95% are embedded in the vacuoles of the yeast cells.

embodiment

Further advantages and the detailed details of the invention are explained below using an exemplary embodiment.

1. Preparation of a gene fusion product

Fusion of two DNA fragments is explained for a first DNA fragment encoding the yeast enzyme invertase, and a second DNA fragment encoding the 76 C-terminal amino acids of a Vicia faba legumin B subunit. The construction should be carried out so that in the gene fusion product to the complete invertase (without translation stop) 3'seitig follow the 76 C-terminal amino acids of the Legumin B subunit.

1.1. Preparation of the starting construct

From an SUC2 gene coding for invertase / 1 /, the first DNA fragment is isolated as an EcoRI / PstI DNA fragment starting 41 base pairs before the start kaston and ending approximately 800 base pairs after the stop codon. It is cloned into an EcoRI / PstI restriction site of a vector plasmid pBS.M 13 "such that the 5 'end of the first DNA fragment is located at the EcoRI restriction site of this vector plasmid. Subunit of Vicia faba is isolated as a second DNA fragment, a Kpnl / Sphl DNA fragment which encodes the region of the C-terminal end, it is in the same vector plasmid and with the same orientation as the first DNA fragment and in a For this purpose, the KpnI restriction site of the legumin B subunit gene as well as the PstI restriction site of the vector plasmid pBS.M 13 "(containing the SUC2 gene) must be pre-clipped of T4 DNA polymerase with blunt ends. In the vector plasmid, the legumin DNA fragment is 3 'side of the SUC2 gene. The starting construct thus produced is then transformed into a bacterial host strain JM101 of Escherichia coli / 3 /. By using a "helper" phage KO7, single-stranded DNA of the vector plasmid containing both DNA fragments is produced therefrom in a customary manner / 4 /.

1.2. Fusion of the two DNA fragments

First, an oligonucleotide having the nucleic acid sequence 5'-CCACCACAAAG 111 IG 111IACTTCCCTTACT-3 'is synthesized.

250ng of the single-stranded DNA according to 1.1. and 30 ng of this (phosphorylated) oligonucleotide are dissolved together in 0.014 ml of water. Then 0.003 ml of 1 molar NaCl and 0.5 molar Tris-HCl with a pH of 7.4 are added. This ar mixture is incubated 10 min at 80 ⁰ C and then for 2 h at 55 ° C.

Thereafter, the batch is cooled to 0 ° C and the following solutions are added:

0.0035 ml of 0.1 molar MgC ^;

0.003 ml of 0.1 molar DTT;

0.002 ml 4-millimolar ATP; 0.002 ml each of 10 millimolar dATP, dGTP, dCTP and dTTP;

0.002 ml of T4 DNA ligase of 1000 U / ml;

0.0005 ml Klenow enzyme of 6,000 U / ml;

0.002 ml of water

This extended approach is incubated at 15 ^° C. for 16 h.

1.3. Selection of the gene fusion product

An aliquot of 0.01 ml of the 1.2. treated batch is transformed into the bacterial host strain JM101, which is streaked on agar medium. The resulting colonies are transferred in a known manner to nitrocellulose filters, which - as also customary - are treated as if a Kolonia-Hybridisier mg to take place. For diesu hybridization, the radioactively-labeled oligonucleotide kinase is used, the reaction is carried out in 5x SSC (10 x SSC = 1.5 M sodium chloride and 0.15M sodium citrate) at 60 ⁰ C.

Bacterial colonies containing a vector plasmid with a built-in oligonucleotide are known to generate signals in autoradiography so that from the signals so determined, the plasmid DNA can be isolated by analysis with appropriate restriction enzymes and the selected clones can be checked by DNA sequence analysis. The details of this are well known, so that can be dispensed with a detailed presentation / 5 /.

2. Expression in yeast

In the following, "yeast" always denotes ° in the host strain of the species Saccharomyces cerevislae.

2.1. Incorporation of the gene fusion product into a yeast expression plasmid

The gene fusion product is now incorporated into a yeast expression plasmid pAAH 5/6 /: an EcoRI / Sph1 DNA fragment containing the gene fusion product and a yeast expression plasmid pAAH5 cut with the restriction gene HindIII are already in provided above with blunt ends and ligated. Of the constructs thus obtained, those are selected as yeast expression vectors whose "SUC 2 gene side" is on the ADH1 promoter and whose "legumin gene side" is on the ADH 1 terminator.

2.2. Transformation into yeast

The transformation into yeast takes place in a way as already described in principle / 7 /. A yeast host strain SEY6210 (alpha-leu 2-3,112 ura 3-52, his 3-Delta200, lys 2-801, sue 2-Delta9 GAU / 8 / in a yeast target medium HVM with 0.5% peptone, 1% glucose and 0.5% yeast extract is cultured until the late logarithmic growth phase of about 10 * cells per ml, then 10 ° cells are centrifuged at 5600 g for 5 min, in 10 mM Tris / 1 mM EDTA with a pH of 8.0 washed and suspended in 0.5 ml of the same batch. After addition of an equal volume of 0.2 M LiCl followed by incubation for 1 h at 3O ⁰ C with shaking 0.001 mg plasmid DNA, dissolved in 0, . nl added 70% polyethylene glycol 4000 and incubated for the last time all 1 hour, also at 30 ° C - There is further incubated for 30 min at 3O ⁰ C. Finally, 0.1 01 ml, added to ¹ Ao volume of this solution.. After heat treatment for 5 min at 42 ° C. and subsequent cooling in an ice bath, the yeast cells are washed twice in distilled water, the pellet is now set to 0, 2ml 1 M sorbitol / 10 mM CaCl ₂ suspended / 33% -HVM, incubated for 1 h at 37 ⁰ C with shaking and ausplsttiert in 0.05 ml aliquots on Hefeminimalmcdium to HMM / 9 / without added leucine. In this way, both the original yeast expression plasmid pAAH5 and a yeast expression vector pAAH 5-N-L76 selected according to the invention can be transformed into the yeast host strain SEY6210. Here, as shown in the following Table 1, the transformation frequency of the yeast expression plasmid pAAH5 is highest.

Tab. 1: Transformation frequencies in yeast

Yeast strain Transformed number of transformation

Plasmid transformant frequency · 10 ~ ⁶

per microgram of DNA

SEY6210 pAAH5 155 1.55

SEY6210 pAAH5-N-L76 59 0.59

For the characterization of the transformed yeast cells obtained, it is important to establish whether a transformed plasmid is present as an autonomously replicating plasmid in a yeast cell or integrated into its chromosome. For this purpose, the mitotic stability is investigated. It is well known that autonomously replicating plasmids are rapidly lost in yeast transformants, especially in nonselective culture, thus mitotically unstable, whereas those yeast transformants in which the plasmid is chromosomally integrated are found to be mitotically stable [10]. The transformed yeast cells are therefore selectively cultivated in liquid HMM selectively or in liquid HVM for at least 16 generations and then plated out on HMM with or without leucine. By comparing the number of yeast colonies to HMM without or with leucine, the mitotic stability can be determined as a percentage.

It turns out that neither the yeast transformant SEY6210 / pAAH 5 nor the yeast transformant SEY6210 / pAAH 5-N-L76 are mitotically stable, especially when grown on nonselective HVM, ie that their respective transformed plasmid is present as an autonomously replicating plasmid in the yeast cells got to.

The result can be confirmed by reverse transformation of the plasmids into a bacterial host strain DH 5 alpha / 3 / of Escherichia coli. For this, the plasmid DNA is isolated from the yeast cells / 11 / and in the host strain DH 5 alpha transfo mized ^r / 3 /. If one then compares the restriction pattern of the plasmid DNA from the transformants with those from the original plasmids, one finds identical match.

2.3. Determination of invertase activity

The invertase activity is determined in the manner already described / 12 /. By using intact yeast cells in culture medium, the secreted amount of the invertase can be determined. By permeabilizing these yeast cells with Triton X100, the total activity can be measured so that the invertase activity retained in the yeast cells can be calculated.

The invertase formed in yeast from the SUC2 gene (original) is secreted by its own N-terminal amino acid signal sequence in Saccharomyces cerevisiae.

If only the 76 C-terminal amino acids in the yeast expression vector pAAH 5-NL 76 are fused behind the gene for invertase by the gene of the legumin B subunit, then 97% or more of the invertase formed is transported by the yeast transformants into the invertase vacuole.

Literature / Sources

1 Taussig, R./Carlson, M., Nucl. Acids Res. 11 (1983), pp. 1943-1954.

2 Manufactured by Stratagene, USA.

3 Hanahan, D., J. Mol. Biol. 166 (1983), p.557-580.

4 Vieira, J., Messing, J., Methods Enzymol. 153 (D) (1987), pp. 3-11.

5 Maniatis, T./Fritsch, E.F. et al., Molecular Cloning, Cold Spring Harbor Lab, New York, 1982.

6 Ammerer, G., in "Methods in Enzymology" 101C (1983), p.192-201.

7 lto, H./Fukada, Y. et al., J. Bacteriol. 15 (1983), pp. 163-168.

8 Bankaitis, V.A./Johnson, LM. et al., Proc. Natl. Acad. Sci. USA83 (1986), pp. 9075-9079.

9 Tanaka, A./Ohnishi, N. et al., J. Ferment.Technol. 45 (1967), p.617-623.

10 Kingsman, A.J. / Clarke, L et al. Gene 7 (1979), pp. 141-152.

11 Case, M.E. in (Hrsgb .: Lurquin, P.F./Leinhof, A.) "Genetic Engineering in eucaryotes", Plenum Press Publ. Corp. 1983, p.1-5.

12 Goldstein, A./Lampen, J.D., Methods Enzymol. 42 (1975), pp. 505-511.

Claims (5)

1. A process for the production of proteins and polypeptides which are incorporated as Resei vestoffe in the cells of a host strain of the species Saccharomyces cerevisiae, wherein the structural gene of the protein or polypeptide is isolated, the protein or polypeptide has at the N-terminal end a secretory amino acid signal sequence which controls the transport into the endoplasmic reticulum, a DNA fragment coding for a vacuolar transport signal sequence is downstream of the DNA fragment in a known manner with the structure of the protein or polypeptide, whereby both DNA fragments are cloned into a vector plasmid which is suitable for the production of single-stranded DNA . the first double-stranded DNA of the vector plasmid is made single-stranded, - Is hybridized to the now single-stranded DNA 30 to 35 base pairs long oligonucleotide whose nucleic acid sequence is selected in dependence on the vacuole transport signal sequence so that an exact fusion of this vacuole transport signal sequence encoding DNA fragment with the for the Protein or polypeptide-encoding DNA fragment takes place, - The thus-formed fusion product is transformed into the host strain, and wherein further - the vacuolar transport signal sequence is obtained from a plant Speicherglobulin, characterized in that the Vakuolen- ^T obtained from the plant-Speicherglobulin ransport signal sequence comprises a C-terminal amino acid sequence of at least 76 amino acids at the C-terminal amino acid sequence of the protein or polypeptide to be incorporated into the cell. 2. The method according to claim 1, characterized in that the vacuole transport signal sequence is obtained from legumin. 3. The method according to claim 1 and 2, characterized in that the amino acid signal sequence from the legumin B subunit of Vicia faba is obtained. 4. The method according to claim 1 to 3, characterized in that the structural gene of a hydrolase or invertase is used. 5. The method according to claim 1 to 4, characterized in that the structural gene of chymosin is used.