Process for bacterial production of polypeptides

Abstract

Refractile particles containing a heterologous polypeptide as an insoluble aggregate are recovered from bacterial periplasm. The process involves culturing bacterial cells so as to express nucleic acid encoding phage lysozyme and nucleic acid encoding the heterologous polypeptide under separate promoters, disrupting the cells mechanically to release the phage lysozyme so as to release retractile particles from the bacterial cellular matrix, and recovering the released retractile particles from the periplasm. Chloroform is not used in any step and the recovery step minimizes co-recovery of cellular debris with the released retractile particles.

Description

RELATED APPLICATIONS [0001] This application is a continuation application and claims the benefit under 35 USC § 120 of co-pending U.S. patent application Ser. No. 09 / 422,528, filed Oct. 21, 1999, which claims the benefit, under 35 USC § 119 of U.S. Provisional Patent Application Ser. No. 60 / 106,053 filed Oct. 28, 1998, the contents of which are incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates to a process for producing and recovering polypeptides from bacterial cells. More particularly, this invention relates to a process wherein recovery of insoluble recombinant polypeptides from bacterial periplasm is increased. [0004] 2. Description of Related Disclosures [0005]Escherichia coli has been widely used for the production of heterologous proteins in the laboratory and industry. E. coli does not generally excrete proteins to the extracellular medium apart from colicins and hemolysin (Pugsley and Schwartz, Micro...

AI Technical Summary

Benefits of technology

[0025] (c) recovering the released refractile particles from the periplasm, whereby chloroform is not used in any step of the process, and wherein the recovery step minimizes co-recovery of cellular debris with the released retractile particles.

[0026] It was found in cell recovery that mechanical breakage by itself is not sufficient for efficient release of the insoluble polypeptide in aggregate form such as refractile particles and that HEW-lysozyme does not work well. Coordinated expression of nucleic acid encoding phage lysozyme with nucleic acid encoding the polypeptide of interest provides a highly effective method for releasing insoluble refractile particles from the entanglement with the peptidoglycan layer. When the phage lysozyme gene is cloned behind a tightly-controlled promoter, for example, the pBAD promoter (also referred to as the ara promoter), cytoplasmic accumulation of phage lysozyme may be induced by the addition of an inducer (such as arabinose) at an appropriate time near the end of fermentation. By placing the nucleic acid expression of heterologous polypeptide and phage lysozyme under separate promoter control, one can independently regulate their production during fermentation. Without a signal sequence, the accumulated phage lysozyme is tightly locked up in the cytoplasmic compartment. Upon mechanical disruption of the cells, phage lysozyme is released to degrade the peptidoglycan layer. Furthermore, the optimal pH for T4-phage-lysozyme activity, which is a preferred embodiment, is about 7.3, which is about the neutral pH of most typical harvest broths.

[0027] The induction of the gene encoding the bacteriophage lysozyme after expression of the nucleic acid encoding the heterologous polypeptide results in a significant increase in the amount of insoluble heterologous polypeptide recovered from the periplasm of bacteria after mechanical cell disruption. The phage lysozyme is trapped in the cytoplasmic compartment during fermentation until release by such disruption. Besides product yield, the success of a recovery process is judged by the ease of operation, the process flow, the turn-around time, as well as the operation cost. The present invention alleviates several if not all these bottlenecks encountered in the large-scale recovery process.

[0028] The process herein also allows use of phage lysozyme at high cell density and increased scale. At high density, even partial leakiness of expression could have disastrous results. Further, it would not be expected that induction at the end of a long fermentation process and after substantial product accumulation would produce enough of the phage lysozyme to be effective. The present process does not pose problems at high cell densities such as increased viscosity and excessive foaming during the fermentation process. The examples herein demonstrate that the process of this invention enables the attainment of high cell density, effective induction and action of the system, and the processing of lysates derived from high-density cultures. Additionally, at least certain embodiments of the process herein require less mechanical disruption of the cells, leading to less large-scale processing time than with conventional processing.

Problems solved by technology

The conventional isolation of heterologous polypeptide from gram-negative bacteria poses problems owing to the tough, rigid cell walls that surround these cells.

However, these chemicals are not inert and may have detrimental effects on many recombinant protein products or subsequent purification procedures.

They do not translate easily and efficiently and are generally unsuitable as large-scale methods.

There are several disadvantages to the use of the HEW-lysozyme addition for isolating periplasmic proteins.

Also, the method is not suitable for lysis of large amounts of cells because the lysozyme addition is inefficient and there is difficulty in dispersing the enzyme throughout a large pellet of cells.

Although these methods have worked on a laboratory scale, they involve too many steps for an efficient large-scale recovery process.

It is believed that these mutants are deficient in some component of the outer bacterial membrane leading to an increase in the cells' permeability.

The results were disappointing in that almost 40% of the total product was lost to the supernatant after three passes through the Gaulin homogenizer.

Product recovery was not significantly improved even when the classical techniques of EDTA and HEW-lysozyme additions were employed.

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