Journal of Industrial Microbiology & Biotechnology (1999) 22 , 176–180  1999 Society for Industrial Microbiology 1367-5435/99/$12.00 http:/ / www.stockton-press.co.uk/ jim Synthesis of mini-proinsulin precursors using N-termini of human TNF  as fusion partners in recombinant Escherichia coli CS Shin 1 , MS Hong 1 , DY Kim 2 , J Lee 2 and Y-H Park 2 1 Bioprocess Engineering Laboratory, Hanhyo Institutes of Technology, Yusong, Taejon 305–390, South Korea; 2 Biochemical Process Engineering RU, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305–600, South Korea Synthesis of human mini-proinsulin precursors was investigated in controlled fed-batch cultures at high cell concen- trations of recombinant Escherichia coli. Transcription of the recombinant gene was controlled by a T7 promoter system. The human mini-proinsulin was prepared by substituting a C-chain peptide of natural proinsulin with a peptide sequence of only nine amino acids. The reduced size of fusion proinsulin and hence the increased purity of human insulin in the recombinant product may contribute to increasing the fermentation yield of human insulin. Three precursors (T1-, T2-, and T3-M2PI) were constructed by utilizing the N-terminus residues of human tumor necrosis factor  as fusion partners. The T2 precursor was most soluble in the cytoplasm, and exerted the most inhibitory effect on recombinant cell growth. In the production of T2-M2PI, significant amounts of undesirable meta- bolic by-products (acetate and ammonia) accumulated in the culture broth even at very low specific cell growth rate. The major portion of all synthesized precursors aggregated to insoluble inclusion bodies but the protein aggre- gates were easily converted to monomers in the presence of the anionic detergent (SDS) without using any reducing agent. With the expression of T1-M2PI, growth inhibition was minimal, and the maximum volumetric yield of mini- proinsulin (M2PI) in fermentation cultures was at the highest level among the synthesized precursors. Keywords: Escherichia coli; mini-proinsulin; N-terminus residues; human tumor necrosis factor ; fusion partners Introduction One potential disadvantage of direct expression of foreign proteins in Escherichia coli cytoplasm is that synthesis of the desired proteins is initiated with methionine which is not efficiently processed in vivo. To overcome this problem, several methods have been developed, including the secretion of heterologous protein [5,6,10,11,15] and enzy- matic removal of an appropriately designed N-terminal fusion partner [3,4,9,14]. Sometimes protein stability is a problem in the production of small heterologous proteins or peptides such as human proinsulin because of their short half-life in the host cell [16]. In order to increase the stab- ility of expressed protein in E. coli, expression in fusion proteins has also been widely employed. Although bacterial intracellular fusion systems are frequently characterized by production of insoluble inclusion bodies [17], the choice of fusion partner sometimes significantly determines the solu- bility, toxicity to host cell, and/or yield (or concentration) of expressed fusion protein, which in turn affects the efficiency of complex downstream processes [2,7,8,12]. At the same time, there has not been the same physiological background for the development of fermentation processes producing the desired recombinant proteins with high stab- ility as well as high yield, where the biomass concentration is increased to high levels (25g L -1 ). Therefore, the Correspondence: Dr J Lee, Biochemical Process Engineering RU, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305–600, South Korea Received 31 August 1998; accepted 22 February 1999 characteristics of the fusion expression system in protein stability, yield, and bacterial physiology need to be investi- gated in detail for development of optimal fermentation processes. Human insulin derived from rDNA technology was the first marketed human health-care product, commercialized by Eli Lilly and Company in the United States in 1982. Since the bulk market of human insulin requires large-scale fermentation (10 000 L) producing human proinsulin, even small improvements in protein concentration signifi- cantly affect productivity of the fermentation process. Therefore, it is of great importance to develop an efficient and stable expression system for a high-yield fermen- tation process. The present investigation is concerned with the synthesis of recombinant fusion human proinsulin in fed-batch cul- tures of E. coli. Human proinsulin was prepared by replac- ing the natural C-chain peptide (35 amino acids) with a new peptide sequence consisting of only nine amino acids. This mutant human proinsulin has been named as ‘mini- proinsulin’ [13]. The fusion mini-proinsulin produced can be converted to authentic human insulin by removing the fusion partner and the pro-sequence by in vitro treatment with cyanogen bromide and proteolytic enzymes, respect- ively [1]. In the present work, the effects of various fusion partners (derived from N-terminus residues of tumor necrosis factor ) on cell growth and gene expression were investigated in well-controlled fed-batch cultures at high cell concentrations. Some important characteristics in solu- bility and aggregation of synthesized recombinant precur- sors were demonstrated.